Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases

ABSTRACT

Compositions and methods are provided for treatment of diseases involving unwanted neovascularization (NV). The invention provides treatments that control NV through selective inhibition of pro-angiogenic biochemical pathways, including inhibition of the VEGF pathway gene expression and inhibition localized at pathological NV tissues. Tissue targeted nanoparticle compositions comprising polymer conjugates and nucleic acid molecules that induce RNA interference (RNAi) are provided. The nanoparticle compositions of the invention can be used alone or in combination with other therapeutic agents such as VEGF pathway antagonists. The compositions and methods can be used for the treatment of NV diseases such as cancer, ocular disease, arthritis, and inflammatory diseases.

This application claims priority from U.S. Provisional PatentApplication No. 60/900,858, filed Feb. 9, 2007. This application is alsoa continuation-in-part of U.S. patent application Ser. No. 11/824,084,filed Jun. 29, 2007, which is a continuation application ofInternational Patent Application No. PCT/US06/13645, filed Apr. 12,2006, which designates the United States and which claims priority under35 U.S.C. §119(e) from U.S. Provisional Patent Application No.60/670,717, filed Apr. 12, 2005. The application also relates to thefollowing applications: International Patent Application No.PCT/US05/03858, filed Feb. 7, 2005; International Patent Application No.PCT/US05/03857, filed Feb. 7, 2005; and International Patent ApplicationNo. PCT/US03/24587, filed Aug. 6, 2003. Each of the disclosures ofPCT/US06/13645, PCT/US05/03858, PCT/US05/03857, PCT/US03/24587, U.S.Ser. No. 11/824,084, U.S. 60/900,858 and U.S. 60/670,717 is herebyincorporated by reference in its entirety.

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is sequence.txt. The text file is 76,968 bytes, wascreated on Feb. 23, 2009 and was submitted electronically via EFS-Web onApr. 1, 2009 in this application.

FIELD OF THE INVENTION

The invention provides compositions and methods for treatments ofdiseases with unwanted neovascularization (NV), often an abnormal orexcessive proliferation and growth of blood vessels. The development ofNV itself often times has adverse consequences or it can be an earlypathological step in disease. Despite introduction of new therapeuticantagonists of angiogenesis, including antagonists of the VEGF pathway,treatment options for controlling NV are inadequate and a large andgrowing unmet clinical need remains for effective treatments of NV,either to inhibit disease progression or to reverse unwantedangiogenesis. Since NV also can be a normal biological process,inhibition of unwanted NV is preferably accomplished with selectivityfor a pathological tissue, which preferably requires selective deliveryof therapeutic molecules to the pathological tissue.

The present invention overcomes this hurdle by providing treatments tocontrol NV through selective inhibition of pro-angiogenic biochemicalpathways, including inhibition of VEGF pathway gene expression andinhibition localized at pathological NV tissues. The present inventionprovides compositions and methods for using a tissue targetednanoparticle composition comprising polymer conjugates and furthercomprising nucleic acid molecules that induce RNA interference (RNAi).The present invention provides compositions and methods for inhibitionof individual genes or combinations of genes active in NV and morepreferably in the VEGF pathway. The dsRNA nanoparticle compositions ofthe invention can be used alone or in combination with other therapeuticagents, including targeted therapeutics, including VEGF pathwayantagonists, such as monoclonal antibodies and small moleculeinhibitors, and targeted therapeutics inhibiting EGF and its receptor orPDGF and its receptors or MEK or Bcr-Abl, and immunotherapy andchemotherapy. The present invention also provides compositions andmethods for the treatment of NV disease in a subject, including cancer,ocular disease, arthritis, and inflammatory diseases.

BACKGROUND OF THE INVENTION

Recent US FDA approved therapeutic agents, including Avastin, andMacugen, provide some benefit for NV diseases. Some of these agents actby binding to and inhibiting the action of Vascular Endothelial GrowthFactor (VEGF), but these agents are not effective for many patients.Other agents being evaluated in clinical studies show signs that theymay provide some benefit by binding to and inhibiting the action of thereceptors for VEGF, or “down stream” proteins used by these receptorsfor signal transduction. The picture that has emerged is that means tocontrol this VEGF “pathway” can provide a level of control of NV thatprovides benefit for some patients. In addition, studies of a series ofsmall molecule kinase inhibitors found that a inhibitor called sunitinibthat has activity against multiple kinase proteins, VEGF receptor, PDGFreceptor, FLT3, and Kit, offers better clinical benefit for NV diseases.However, these benefits are still inadequate for most patients andbetter therapeutic means to control the VEGF pathway still are needed.The agents developed to date are mostly antagonists of VEGF or itsreceptors, VEGF R1 and VEGF R2. One problem that has emerged with use ofantagonists appears to be a response by the pathological tissues toincrease production of VEGF. Thus an attractive means to improvetherapeutic control of NV is to inhibit production of the VEGF pathwayproteins, i.e., down regulate their gene expression, and doing so byinducing RNA interference through in vivo delivery of small interferingdsRNA oligonucleotides (siRNA).

RNA interference (RNAi) is a post-transcriptional process where a doublestranded RNA inhibits gene expression in a sequence specific fashion.The RNAi process occurs in at least two steps: During one step, a longdsRNA is cleaved by an endogenous ribonuclease into shorter, 21- or23-nucleotide-long dsRNAs. In another second step, the smaller dsRNAmediates the degradation of an mRNA molecule with a matching sequenceand as a result selectively down regulating expression of that gene.This RNAi effect can be achieved by introduction of either longerdouble-stranded RNA (dsRNA) or shorter small interfering RNA (siRNA) tothe target sequence within cells. Recently, it was demonstrated thatRNAi can also be achieved by introducing of plasmid that generate dsRNAcomplementary to target gene.

RNAi methods have been successfully used in gene function determinationexperiments in Drosophila ^((20, 22, 23, 25)) , C. elegans^((14, 15, 16)), and Zebrafish⁽²⁰⁾. In those model organisms, it hasbeen reported that both the chemically synthesized shorter siRNA or invitro transcribed longer dsRNA can effectively inhibit target geneexpression. Methods have been reported that successfully achieved RNAieffects in non human mammalian and human cell cultures⁽³⁹⁻⁵⁶⁾. However,RNAi effects have been difficult to observe in adult animal models⁽⁵⁷⁾.This is for several reasons including: introduction of a longdouble-stranded RNA into mammalian cells can trigger an antiviral immuneresponse including up-regulation of interferon, resulting in apoptosisand death of the cells that can be either detrimental or beneficial tothe desired therapeutic effect: the efficiency of dsRNA entry into thetarget cell is low, especially in animals; short dsRNA molecules arerapidly excreted from the blood into the urine; and RNA molecules can bedegraded by RNAse nuclease activity. Although RNAi has potentialapplications in both gene target validation and nucleic acidtherapeutics, progress of the technology has been hindered due to thepoor delivery of RNAi molecules into animal disease models.

It is apparent, therefore, that improved methods for delivering RNAimolecules in vivo are of great importance. It is also apparent thattissue targeted delivery of nucleic acid molecules inducing RNAi are ofgreat importance. It is also apparent that methods for deliveringnucleic acid molecules inducing RNAi selective for VEGF pathway geneswill be of great benefit for the treatment of NV diseases. These needsare addressed by the compositions and methods of the invention.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to utilize RNAi tomodulate angiogenesis process in order to reverse the disease process bydown regulating gene expression involved in NV pathogenesis, morespecifically genes in the VEGF pathway.

It is therefore an object of the invention to provide compositions andmethods for inhibiting expression of one or more VEGF pathway genes in amammal. It is a further object of the invention to provide compositionsand methods for treating NV disease by inhibiting expression of one ormore VEGF pathway genes alone or in combination with other agentsincluding antagonists of the same VEGF pathway.

In achieving these objects there have been provided compositions andmethods for down regulating endogenous VEGF pathway genes, comprisingadministering to a tissue of the mammal a composition comprising adouble-stranded RNA molecule where the RNA molecule specifically reducesor inhibits expression of the endogenous VEGF pathway gene. This downregulation of an endogenous gene may be used for treating a disease thatis caused or exacerbated by activity of the VEGF pathway. The diseasemay be in a human.

Additional embodiments of the invention have been provided as follows:

-   Paragraph 1. A nucleic acid molecule selected from the group    consisting of:

(a) 5′-CCUGAUGAGAUCGAGUACAUCUUCA-3′ (b) 5′-GAGAGAUGAGCUUCCUACAGCACAA-3′(c) 5′-CACAACAAAUGUGAAUGCAGACCAA-3′ (d) 5′-GAGUCCAACAUCACCAUGCAGAUUA-3′(e) 5′-AGUCCAACAUCACCAUGCAGAUUAU-3′ (f) 5′-CCAACAUCACCAUGCAGAUUAUGCG-3′(g) 5′-CACCAUGCAGAUUAUGCGGAUCAAA-3′; and(h) 5′-GCACAUAGGAGAGAUGAGCUUCCUA-3′.

-   Paragraph 2. A 25 base pair nucleic acid molecule that is the    complement of a nucleic acid molecule of paragraph 1.-   Paragraph 3. A 25 base pair, blunt-ended double stranded nucleic    acid selected from the group consisting of:

(a) Sense strand: 5′-CCUGAUGAGAUCGAGUACAUCUUCA-3′ and antisense strand:5′-UGAAGAUGUACUCGAUCUCAUCAGG-3′; (b) Sense strand:5′-GAGAGAUGAGCUUCCUACAGCACAA-3′ and antisense strand:5′-UUGUGCUGUAGGAAGCUCAUCUCUC-3′; and (c) Sense strand:5′-CACAACAAAUGUGAAUGCAGACCAA-3′ and antisense strand:5′-UUGGUCUGCAUUCACAUUUGUUGUG-3′.

-   Paragraph 4. A composition comprising the nucleic acid molecule of    paragraph 2 or the double-stranded nucleic acid molecule of    paragraph 3 and a pharmaceutically acceptable carrier.-   Paragraph 5. The composition of paragraph 4, further comprising one    or more additional nucleic acid molecules that induce RNA    interference and decrease the expression of a gene of interest.-   Paragraph 6. The composition of paragraph 5, wherein the one or more    additional nucleic acid molecules decrease the expression of human    VEGF.-   Paragraph 7. The composition of paragraph 5, wherein the one or more    additional nucleic acid molecules decrease the expression of a    VEGF-pathway gene other than VEGF.-   Paragraph 8. The composition of paragraph 5, wherein at least one of    the one or more additional nucleic acid molecules decreases the    expression of a gene that promotes angiogenesis.-   Paragraph 9. The composition of paragraph 5, wherein at least one of    the one or more additional nucleic acid molecules decreases the    expression of a gene selected from the group consisting of; VEGFR1,    VEGFR2, VEGFR3, PDGF, PDGFR-α, PDGFR-β, EGF, EGFR, RAF-a, RAF-c,    AKT, RAS, NFkB, HIF, bFGF, bFGFR, Her-2, c-Met, c-Myc and HGF.-   Paragraph 10. A composition comprising the nucleic acid molecule of    paragraph 2 or the double stranded nucleic acid molecule of    paragraph 3, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGFR1.-   Paragraph 11. A composition comprising the nucleic acid molecule of    paragraph 2 or the double stranded nucleic acid molecule of    paragraph 3, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGFR2.-   Paragraph 12. A composition comprising:

(a) the nucleic acid molecule of paragraph 2 or the double strandednucleic acid molecule of paragraph 3,

(b) a nucleic acid molecule that induces RNA interference and decreasesthe expression of human VEGFR1; and

(c) a nucleic acid molecule that induces RNA interference and decreasesthe expression of human VEGFR2.

-   Paragraph 13. The composition of paragraph 4, wherein the carrier is    a nucleic acid delivery vehicle.-   Paragraph 14. The composition of paragraph 13, wherein the nucleic    acid delivery vehicle is synthetic.-   Paragraph 15. The composition of paragraph 14 wherein the synthetic    nucleic acid delivery vehicle comprises a core region comprising a    cationic polymer and the nucleic acid.-   Paragraph 16. The composition of paragraph 15, wherein the cationic    polymer is polyethyleneimine.-   Paragraph 17. The composition of paragraph 15, wherein the cationic    polymer is a histidine-lysine co-polymer.-   Paragraph 18. The composition of paragraph 15, wherein the synthetic    nucleic acid delivery vehicle further comprises a protective    hydrophilic layer.-   Paragraph 19. The composition of paragraph 18, wherein the    protective hydrophilic layer comprises a component selected from the    group consisting of: polyethylene glycol, a polyacetal and a    polyoxazoline.-   Paragraph 20. The composition of paragraph 15, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 21. The composition of paragraph 18, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 22. The composition of paragraph 20, wherein the targeting    moiety binds a tumor specific molecule or an angiogenesis-specific    molecule.-   Paragraph 23. The composition of paragraph 20, wherein the targeting    moiety binds endothelial cells.-   Paragraph 24. The composition of paragraph 23, wherein the targeting    moiety binds an integrin on vascular endothelial cells.-   Paragraph 25. The composition of paragraph 24, wherein the targeting    moiety is a peptide comprising the amino acid sequence RGD.-   Paragraph 26. The composition of paragraph 25, wherein the peptide    comprising RGD is a cyclic peptide.-   Paragraph 27. The composition of paragraph 14, wherein the synthetic    nucleic acid delivery vehicle comprises:

(a) a core region comprising PEI;

(b) a protective hydrophilic layer comprising PEG; and

(c) a cyclic RGD-containing peptide targeting moiety.

-   Paragraph 28. The composition of paragraph 4 comprising an    additional therapeutic agent selected from the group consisting of:    an anti-cancer agent, an anti-inflammatory agent and an    anti-infective agent.-   Paragraph 29. The composition of paragraph 4 comprising an    anti-angiogenic agent.-   Paragraph 30. The composition of paragraph 29, wherein the    anti-angiogenic agent is an inhibitor selected from the group    consisting of: an inhibitor of human VEGF, an inhibitor of human    VEGFR1 and an inhibitor of human VEGFR2.-   Paragraph 31. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    a composition selected from the group consisting of:

(a) the nucleic acid of paragraph 2; and

(b) the double stranded nucleic acid of paragraph 3.

-   Paragraph 32. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 4.-   Paragraph 33. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 12.-   Paragraph 34. A method of reducing tumor growth in a subject in need    thereof, comprising the step of administering the nucleic acid of    paragraph 3 or the double stranded nucleic acid of paragraph 41 to    the subject.-   Paragraph 35. A method for decreasing the VEGF protein level in a    cell comprising introducing into the cell the nucleic acid molecule    of paragraph 2 or the double-stranded nucleic acid of paragraph 3.-   Paragraph 36. The nucleic acid molecule of paragraph 1, wherein said    nucleic acid molecule is RNA.-   Paragraph 37. The double-stranded nucleic acid molecule of paragraph    3, wherein said double-stranded nucleic acid molecule is RNA.-   Paragraph 38. A nucleic acid molecule selected from the group    consisting of:

(a) 5′-GCCAACAUAUUCUACAGUGUUCUUA-3′;(b) 5′-CCCUCGCCGGAAGUUGUAUGGUUAA-3′;(c) 5′-CCUCAAGAGCAAACGUGACUUAUUU-3′;(d) 5′-CAAAGGACUUUAUACUUGUCGUGUA-3′;(e) 5′-CAUCACUCAGCGCAUGGCAAUAAUA-3′;(f) 5′-CCACCACUUUAGACUGUCAUGCUAA-3′;(g) 5′-CGGACAAGUCUAAUCUGGAGCUGAU-3′;(h) 5′-UGACCCACAUUGGCCACCAUCUGAA-3′;(i) 5′-GAGGGCCUCUGAUGGUGAUUGUUGA-3′;(j) 5′-CGAGCUCCGGCUUUCAGGAAGAUAA-3′;(k) 5′-CAAUCAAUGCCAUACUGACAGGAAA-3′; and(l) 5′-GAAAGUAUUUCAGCUCCGAAGUUUA-3′.

-   Paragraph 39. A 25 base pair nucleic acid molecule that is the    complement of a nucleic acid molecule of paragraph 39.-   Paragraph 40. A 25 base pair, blunt-ended double stranded nucleic    acid selected from the group consisting of:

(a) Sense strand: 5′-GCCAACAUAUUCUACAGUGUUCUUA-3′ and antisense strand:5′-UAAGAACACUGUAGAAUAUGUUGGC-3′; (b) Sense strand:5′-CCCUCGCCGGAAGUUGUAUGGUUAA-3′ and antisense strand:5′-UUAACCAUACAACUUCCGGCGAGGG-3′; and (c) Sense strand:5′-CCUCAAGAGCAAACGUGACUUAUUU-3′ and antisense strand:5′-AAAUAAGUCACGUUUGCUCUUGAGG-3′.

-   Paragraph 41. A composition comprising the nucleic acid molecule of    paragraph 39 or the double-stranded nucleic acid molecule of    paragraph 40 and a pharmaceutically acceptable carrier.-   Paragraph 42. The composition of paragraph 41, further comprising    one or more additional nucleic acid molecules that induce RNA    interference and decrease the expression of a gene of interest.-   Paragraph 43. The composition of paragraph 42, wherein the one or    more additional nucleic acid molecules decrease the expression of    human VEGFR1.-   Paragraph 44. The composition of paragraph 42, wherein the one or    more additional nucleic acid molecules decrease the expression of a    VEGF-pathway gene other than VEGFR1.-   Paragraph 45. The composition of paragraph 42, wherein at least one    of the one or more additional nucleic acid molecules decreases the    expression of a gene that promotes angiogenesis.-   Paragraph 46. The composition of paragraph 42, wherein at least one    of the one or more additional nucleic acid molecules decreases the    expression of a gene selected from the group consisting of: VEGF,    VEGFR2, VEGFR3, PDGF, PDGFR-α, PDGFR-β, EGF, EGFR, RAF-a, RAF-c,    AKT, RAS, NFkB, HIF, bFGF, bFGFR, Her-2, c-Met, c-Myc and HGF.-   Paragraph 47. A composition comprising the nucleic acid molecule of    paragraph 39 or the double stranded nucleic acid molecule of    paragraph 40, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGF.-   Paragraph 48. A composition comprising the nucleic acid molecule of    paragraph 39 or the double stranded nucleic acid molecule of    paragraph 40, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGFR2.-   Paragraph 49. A composition comprising:

(a) the nucleic acid molecule of paragraph 39 or the double strandednucleic acid molecule of paragraph 40,

(b) a nucleic acid molecule that induces RNA interference and decreasesthe expression of human VEGF; and

(c) a nucleic acid molecule that induces RNA interference and decreasesthe expression of human VEGFR2.

-   Paragraph 50. The composition of paragraph 41, wherein the carrier    is a nucleic acid delivery vehicle.-   Paragraph 51. The composition of paragraph 50, wherein the nucleic    acid delivery vehicle is synthetic.-   Paragraph 52. The composition of paragraph 51, wherein the synthetic    nucleic acid delivery vehicle comprises a core region comprising a    cationic polymer and the nucleic acid.-   Paragraph 53. The composition of paragraph 52, wherein the cationic    polymer is polyethyleneimine.-   Paragraph 54. The composition of paragraph 52, wherein the cationic    polymer is a histidine-lysine co-polymer.-   Paragraph 55. The composition of paragraph 52, wherein the synthetic    nucleic acid delivery vehicle further comprises a protective    hydrophilic layer.-   Paragraph 56. The composition of paragraph 55, wherein the    protective hydrophilic layer comprises a component selected from the    group consisting of: polyethylene glycol, a polyacetal and a    polyoxazoline.-   Paragraph 57. The composition of paragraph 52, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 58. The composition of paragraph 55, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 59. The composition of paragraph 57, wherein the targeting    moiety binds a tumor specific molecule or an angiogenesis-specific    molecule.-   Paragraph 60. The composition of paragraph 57, wherein the targeting    moiety binds endothelial cells.-   Paragraph 61. The composition of paragraph 60, wherein the targeting    moiety binds an integrin on vascular endothelial cells.-   Paragraph 62. The composition of paragraph 61, wherein the targeting    moiety is a peptide comprising the amino acid sequence RGD.-   Paragraph 63. The composition of paragraph 62, wherein the peptide    comprising RGD is a cyclic peptide.-   Paragraph 64. The composition of paragraph 51, wherein the synthetic    nucleic acid delivery vehicle comprises:

(a) a core region comprising PEI;

(b) a protective hydrophilic layer comprising PEG; and

(c) a cyclic RGD-containing peptide targeting moiety.

-   Paragraph 65. The composition of paragraph 41 comprising an    additional therapeutic agent selected from the group consisting of:    an anti-cancer agent, an anti-inflammatory agent and an    anti-infective agent.-   Paragraph 66. The composition of paragraph 41 comprising an    anti-angiogenic agent.-   Paragraph 67. The composition of paragraph 66, wherein the    anti-angiogenic agent is an inhibitor selected from the group    consisting of: an inhibitor of human VEGF, an inhibitor of human    VEGFR1 and an inhibitor of human VEGFR2.-   Paragraph 68. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    a composition selected from the group consisting of:

(a) the nucleic acid of paragraph 39; and

(b) the double stranded nucleic acid of paragraph 40.

-   Paragraph 69. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 41.-   Paragraph 70. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 49.-   Paragraph 71. A method of reducing tumor growth in a subject in need    thereof, comprising the step of administering the nucleic acid of    paragraph 39 or the double stranded nucleic acid of paragraph 40 to    the subject.-   Paragraph 72. A method for decreasing the VEGFR1 protein level in a    cell comprising introducing into the cell the nucleic acid molecule    of paragraph 39 or the double-stranded nucleic acid of paragraph 40.-   Paragraph 73. The nucleic acid molecule of paragraph 38, wherein    said nucleic acid molecule is RNA.-   Paragraph 74. The double-stranded nucleic acid molecule of paragraph    40, wherein said double-stranded nucleic acid molecule is RNA.-   Paragraph 75. A nucleic acid molecule selected from the group    consisting of:

(a) 5′-CCUCUUCUGUAAGACACUCACAAUU-3′;(b) 5′-CCCUUGAGUCCAAUCACACAAUUAA-3′;(c) 5′-CCAAGUGAUUGAAGCAGAUGCCUUU-3′;(d) 5′-CCUCGGUCAUUUAUGUCUAUGUUCA-3′;(e) 5′-CAGAUCUCCAUUUAUUGCUUCUGUU-3′;(f) 5′-GACCAACAUGGAGUCGUGUACAUUA-3′;(g) 5′-CCCUUGAGUCCAAUCACACAAUUAA-3′;(h) 5′-CCAUGUUCUUCUGGCUACUUCUUGU-3′;(i) 5′-UCAUUCAUAUUGGUCACCAUCUCAA-3′;(j) 5′-GAGUUCUUGGCAUCGCGAAAGUGUA-3′;(k) 5′-CAGCAGGAAUCAGUCAGUAUCUGCA-3′;(l) 5′-CAGUGGUAUGGUUCUUGCCUCAGAA-3′; and(m) 5′-CCACACUGAGCUCUCCUCCUGUUUA-3′.

-   Paragraph 76. A 25 base pair nucleic acid molecule that is the    complement of a nucleic acid molecule of paragraph 75.-   Paragraph 77. A 25 base pair, blunt-ended double stranded nucleic    acid selected from the group consisting of:

(a) Sense strand 5′-CCUCUUCUGUAAGACACUCACAAUU-3′ and antisense strand:5′-AAUUGUGAGUGUCUUACAGAAGAGG-3′; (b) Sense strand:5′-CCCUUGAGUCCAAUCACACAAUUAA-3′ and antisense strand:5′-UUAAUUGUGUGAUUGGACUCAAGGG-3′ (c) Sense strand:5′-CCAAGUGAUUGAAGCAGAUGCCUUU-3′ and antisense strand:5′-AAAGGCAUCUGCUUCAAUCACUUGG-3′.

-   Paragraph 78. A composition comprising the nucleic acid molecule of    paragraph 76 or the double-stranded nucleic acid molecule of    paragraph 77 and a pharmaceutically acceptable carrier.-   Paragraph 79. The composition of paragraph 78, further comprising    one or more additional nucleic acid molecules that induce RNA    interference and decrease the expression of a gene of interest.-   Paragraph 80. The composition of paragraph 79, wherein the one or    more additional nucleic acid molecules decrease the expression of    human VEGFR2.-   Paragraph 81. The composition of paragraph 79, wherein the one or    more additional nucleic acid molecules decrease the expression of a    VEGF-pathway gene other than VEGFR2.-   Paragraph 82. The composition of paragraph 79, wherein at least one    of the one or more additional nucleic acid molecules decreases the    expression of a gene that promotes angiogenesis.-   Paragraph 83. The composition of paragraph 79, wherein at least one    of the one or more additional nucleic acid molecules decreases the    expression of a gene selected from the group consisting of; VEGFR1,    VEGF, VEGFR3, PDGF, PDGFR-α, PDGFR-β, EGF, EGFR, RAF-a, RAF-c, AKT,    RAS, NFkB, HIF, bFGF, bFGFR, Her-2, c-Met, c-Myc and HGF.-   Paragraph 84. A composition comprising the nucleic acid molecule of    paragraph 76 or the double stranded nucleic acid molecule of    paragraph 77, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGF.-   Paragraph 85. A composition comprising the nucleic acid molecule of    paragraph 76 or the double stranded nucleic acid molecule of    paragraph 77, and an additional nucleic acid molecule, wherein the    additional nucleic acid molecule induces RNA interference and    decreases the expression of human VEGFR1.-   Paragraph 86. A composition comprising:    -   (a) the nucleic acid molecule of paragraph 76 or the double        stranded nucleic acid molecule of paragraph 77,    -   (b) a nucleic acid molecule that induces RNA interference and        decreases the expression of human VEGFR1; and    -   (c) a nucleic acid molecule that induces RNA interference and        decreases the expression of human VEGF.-   Paragraph 87. The composition of paragraph 78, wherein the carrier    is a nucleic acid delivery vehicle.-   Paragraph 88. The composition of paragraph 87, wherein the nucleic    acid delivery vehicle is synthetic.-   Paragraph 89. The composition of paragraph 88 wherein the synthetic    nucleic acid delivery vehicle comprises a core region comprising a    cationic polymer and the nucleic acid.-   Paragraph 90. The composition of paragraph 89, wherein the cationic    polymer is polyethyleneimine.-   Paragraph 91. The composition of paragraph 89, wherein the cationic    polymer is a histidine-lysine co-polymer.-   Paragraph 92. The composition of paragraph 89, wherein the synthetic    nucleic acid delivery vehicle further comprises a protective    hydrophilic layer.-   Paragraph 93. The composition of paragraph 92, wherein the    protective hydrophilic layer comprises a component selected from the    group consisting of: polyethylene glycol, a polyacetal and a    polyoxazoline.-   Paragraph 94. The composition of paragraph 89, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 95. The composition of paragraph 92, wherein the synthetic    nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 96. The composition of paragraph 94, wherein the targeting    moiety binds a tumor specific molecule or an angiogenesis-specific    molecule.-   Paragraph 97. The composition of paragraph 94, wherein the targeting    moiety binds endothelial cells.-   Paragraph 98. The composition of paragraph 97, wherein the targeting    moiety binds an integrin on vascular endothelial cells.-   Paragraph 99. The composition of paragraph 98, wherein the targeting    moiety is a peptide comprising the amino acid sequence RGD.-   Paragraph 100. The composition of paragraph 99, wherein the peptide    comprising RGD is a cyclic peptide.-   Paragraph 101. The composition of paragraph 88, wherein the    synthetic nucleic acid delivery vehicle comprises:

(a) a core region comprising PEI;

(b) a protective hydrophilic layer comprising PEG; and

(c) a cyclic RGD-containing peptide targeting moiety.

-   Paragraph 102. The composition of paragraph 78 comprising an    additional therapeutic agent selected from the group consisting of:    an anti-cancer agent, an anti-inflammatory agent and an    anti-infective agent.-   Paragraph 103. The composition of paragraph 78 comprising an    anti-angiogenic agent.-   Paragraph 104. The composition of paragraph 103, wherein the    anti-angiogenic agent is an inhibitor selected from the group    consisting of: an inhibitor of human VEGF, an inhibitor of human    VEGFR1 and an inhibitor of human VEGFR2.-   Paragraph 105. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    a composition selected from the group consisting of:

(a) the nucleic acid of paragraph 76; and

(b) the double stranded nucleic acid of paragraph 77.

-   Paragraph 106. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 78.-   Paragraph 107. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 86.-   Paragraph 108. A method of reducing tumor growth in a subject in    need thereof, comprising the step of administering the nucleic acid    of paragraph 76 or the double stranded nucleic acid of paragraph 77    to the subject.-   Paragraph 109. A method for decreasing the VEGFR2 protein level in a    cell comprising introducing into the cell the nucleic acid molecule    of paragraph 76 or the double-stranded nucleic acid of paragraph 77.-   Paragraph 110. The nucleic acid molecule of paragraph 75, wherein    said nucleic acid molecule is RNA.-   Paragraph 111. The double-stranded nucleic acid molecule of    paragraph 77, wherein said double-stranded nucleic acid molecule is    RNA.-   Paragraph 112. A composition selected from the group consisting of:-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 9 a siRNA identified by SEQ ID NO. 14 and a    pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 14 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    14 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    16 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    18 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 14 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 14 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    14 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    16 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    18 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 14 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 9, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 14 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 16 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 11, a siRNA identified by SEQ ID NO. 18 and    a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    14 and a pharmaceutically acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    16 and a pharmaceutically acceptable carrier; and-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    with the sequence of SEQ ID NO. 21, a siRNA identified by SEQ ID NO.    18 and a pharmaceutically acceptable carrier.-   Paragraph 113. A composition selected from the group consisting of:-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 9, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 11, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    with the sequence of SEQ ID NO. 21, and a pharmaceutically    acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 9, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 11, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    with the sequence of SEQ ID NO. 21, and a pharmaceutically    acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 9, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 11, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    with the sequence of SEQ ID NO. 21, and a pharmaceutically    acceptable carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 14, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 2, a siRNA    identified by SEQ ID NO. 16, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 14, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 4, a siRNA    identified by SEQ ID NO. 16, and a pharmaceutically acceptable    carrier;-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 14, and a pharmaceutically acceptable    carrier; and-   a composition comprising a siRNA identified by SEQ ID NO. 6, a siRNA    identified by SEQ ID NO. 16, and a pharmaceutically acceptable    carrier.-   Paragraph 114. The composition of paragraph 112 or 113, further    comprising one or more additional nucleic acid molecules that induce    RNA interference and decrease the expression of a gene of interest.-   Paragraph 115. The composition of paragraph 114, wherein at least    one of the one or more additional nucleic acid molecules decreases    the expression of a gene that promotes angiogenesis.-   Paragraph 116. The composition of paragraph 114, wherein at least    one of the one or more additional nucleic acid molecules decreases    the expression of a gene selected from the group consisting of;    VEGFR1, VEGFR2, VEGFR3, PDGF, PDGFR-α, PDGFR-β, EGF, EGFR, RAF-a,    RAF-c, AKT, RAS, NFkB, HIF, bFGF, bFGFR, Her-2, c-Met, c-Myc and    HGF.-   Paragraph 117. The composition of paragraph 112 or 113, wherein the    carrier is a nucleic acid delivery vehicle.-   Paragraph 118. The composition of paragraph 117, wherein the nucleic    acid delivery vehicle is synthetic.-   Paragraph 119. The composition of paragraph 118 wherein the    synthetic nucleic acid delivery vehicle comprises a core region    comprising a cationic polymer and the nucleic acid.-   Paragraph 120. The composition of paragraph 119, wherein the    cationic polymer is polyethyleneimine.-   Paragraph 121. The composition of paragraph 119, wherein the    cationic polymer is a histidine-lysine co-polymer.-   Paragraph 122. The composition of paragraph 119, wherein the    synthetic nucleic acid delivery vehicle further comprises a    protective hydrophilic layer.-   Paragraph 123. The composition of paragraph 122, wherein the    protective hydrophilic layer comprises a component selected from the    group consisting of: polyethylene glycol, a polyacetal and a    polyoxazoline.-   Paragraph 124. The composition of paragraph 119, wherein the    synthetic nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 125. The composition of paragraph 122, wherein the    synthetic nucleic acid vehicle further comprises a targeting moiety.-   Paragraph 126. The composition of paragraph 124, wherein the    targeting moiety binds a tumor specific molecule or an    angiogenesis-specific molecule.-   Paragraph 127. The composition of paragraph 122, wherein the    targeting moiety binds endothelial cells.-   Paragraph 128. The composition of paragraph 127, wherein the    targeting moiety binds an integrin on vascular endothelial cells.-   Paragraph 129. The composition of paragraph 128, wherein the    targeting moiety is a peptide comprising the amino acid sequence    RGD.-   Paragraph 130. The composition of paragraph 129, wherein the peptide    comprising RGD is a cyclic peptide.-   Paragraph 131. The composition of paragraph 128, wherein the    synthetic nucleic acid delivery vehicle comprises:

(a) a core region comprising PEI;

(b) a protective hydrophilic layer comprising PEG; and

(c) a cyclic RGD-containing peptide targeting moiety.

-   Paragraph 132. The composition of paragraph 112 or 113 comprising an    additional therapeutic agent selected from the group consisting of:    an anti-cancer agent, an anti-inflammatory agent and an    anti-infective agent.-   Paragraph 133. The composition of paragraph 112 or 113 comprising an    anti-angiogenic agent.-   Paragraph 134. The composition of paragraph 133, wherein the    anti-angiogenic agent is an inhibitor selected from the group    consisting of: an inhibitor of human VEGF, an inhibitor of human    VEGFR1 and an inhibitor of human VEGFR2.-   Paragraph 135. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 112 or 113.-   Paragraph 136. A method for reducing neovascularization in a subject    in need thereof comprising the step of administering to the subject    the composition of paragraph 131.-   Paragraph 137. A method of reducing tumor growth in a subject in    need thereof, comprising the step of administering to the subject    the composition of paragraph 112 or 113.-   Paragraph 138. A method of reducing tumor growth in a subject in    need thereof, comprising the step of administering to the subject    the composition of paragraph 131.-   Paragraph 139. A method for decreasing one or more of VEGF, VEGFR1    or VEGFR2 protein levels in a cell comprising introducing into the    cell the composition of paragraph 112.-   Paragraph 140. A method for decreasing the protein level of VEGF or    VEGFR1, or of both VEGF and VEGFR1, in a cell comprising introducing    into the cell the composition of paragraph 113.-   Paragraph 141. A method for decreasing the protein level of VEGF or    VEGFR2, or of both VEGF and VEGFR2, in a cell comprising introducing    into the cell the composition of paragraph 113.

There also has been provided a method for treating a disease in a mammalassociated with undesirable expression of a VEGF pathway gene,comprising applying a nucleic acid composition comprising a dsRNAoligonucleotide, as the active pharmaceutical ingredient (API),associated with a formulation, wherein the formulation can comprise apolymer, where the nucleic acid composition is capable of reducingexpression of the VEGF pathway genes and inhibiting NV in the disease.The disease may be cancer or a precancerous growth and the tissue maybe, for example, a kidney tissue, breast tissue, colon tissue, aprostate tissue, a lung tissue or an ovarian tissue.

As used herein, “oligonucleotides” and similar terms based on thisrelate to short oligos composed of naturally occurring nucleotides aswell as to oligos composed of synthetic or modified nucleotides.Oligonucleotides may be 10 or more nucleotides in length, or 15, or 16,or 17, or 18, or 19, or 20 or more nucleotides in length, or 21, or 22,or 23, or 24 or more nucleotides in length, or 25, or 26, or 27, or 28or 29, or 30 or more nucleotides in length, 35 or more, 40 or more, 45or more, up to about 50, nucleotides in length.

An oligonucleotide that is an siRNA may have any number of nucleotidesbetween 15 and 30 nucleotides. In many embodiments an siRNA may have anynumber of nucleotides between 19 and 27 nucleotides.

In many embodiments, an siRNA may have two blunt ends, or two stickyends, or one blunt end with one sticky end. The overhang nucleotides ofa sticky end can range from one to four nucleotides or more.

In a preferred embodiment, the invention provides siRNA of 25 base pairswith blunt ends.

The terms “polynucleotide” and “oligonucleotide” are used synonymouslyherein.

The composition may further comprise a polymeric carrier. The polymericcarrier may comprise a cationic polymer that binds to the RNA moleculeand forms nanoparticles. The cationic polymer may be an amino acidcopolymer, containing, for example, histidine and lysine residues. Thepolymer may comprise a branched polymer. The composition may comprise atargeted synthetic vector. The synthetic vector may comprise a cationicpolymer as a nucleic acid carrier, a hydrophilic polymer as a stericprotective material, and a targeting ligand as a target cell selectiveagent. The polymer may comprise a polyethyleneimine or apolyhistidine-lysine copolymer or a polylysine modified chemically orother effective polycationic carriers that can be used as the nucleicacid carrier module, the hydrophilic polymer may comprise a polyethyleneglycol or a polyacetal or a polyoxazoline, and the targeting ligand maycomprise a peptide comprising an RGD sequence or a sugar or a sugaranalogue or an mAb or a fragment of an mAb, or any other effectivetargeting moieties.

In any of these methods, an electric field may be applied to a tissuesubstantially contemporaneously with the composition or subsequent toapplication of the composition. The composition and method of theinvention comprises dsRNA oligonucleotides with a sequence matching anendogenous VEGF pathway gene or a mutated endogenous gene, and at leastone mutation in the mutated gene may be in a coding or regulatory regionof the gene. In any of these methods, the endogenous gene may beselected from the group consisting of VEGF pathway genes includinggrowth factor genes, protein serine/threonine kinase genes, proteintyrosine kinase genes, protein serine/threonine phosphatase genes,protein tyrosine phosphatase genes, receptor genes, and transcriptionfactor genes. The selected gene may include one or more genes from thegroup consisting of VEGF, VEGF-R1, VEGF-R2, VEGF-R3, VEGF121, VEGF165,VEGF 189, VEGF206, RAF-a, RAF-c, AKT, Ras, NF-Kb. The selected gene mayinclude one or more genes from other biochemical pathways associatedwith NV including HIF, EGF, EGFr, bFGF, bFGFr, PDGF, and PDGFr. Theselected gene may include one or more genes from other biochemicalpathways operative in concert with NV including Her-2, c-Met, c-Myc andHGF.

The present invention also provides compositions and methods for nucleicacid agents inducing RNAi to inhibit multiple genes including cocktailsof siRNA (siRNA-OC). The compositions and methods of the invention mayinhibit multiple genes substantially contemporaneously or they mayinhibit multiple genes sequentially. In a preferred embodiment siRNA-OCagents inhibit three VEGF pathway genes: VEGF, VEGF receptor 1, and VEGFreceptor 2. In another preferred embodiment siRNA-OC are administeredsubstantially contemporaneously. The present invention provides agentswith gene inhibition selectivity derived from matching the sequence ofthe siRNA largely to a sequence in the targeted gene mRNA. It alsoprovides siRNA agents with substantially similar physiochemicalproperties that inhibit different genes in the VEGF pathway. It alsoprovides nanoparticle compositions largely independent of the siRNAsequence. It also provides methods for treatment of human diseases,especially NV related diseases, which can be treated with inhibitors ofmultiple endogenous genes. It also provides methods for treatment ofhuman diseases by combinations of therapeutic agents administeredsubstantially contemporaneously in some cases and, in other cases,sequentially.

One aspect of the present invention provides compositions and methodsfor treatment of cancer, arthritis, blindness, infectious diseases andinflammatory diseases. In another aspect of the present inventionnucleic acid agents inducing RNAi are used in concert with othertherapeutic agents, such as but not limited to small molecules andmonoclonal antibodies (mAb), in the same therapeutic regimen.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of DLD-1 colon xenograft tumor growth byVEGF pathway siRNA nanoparticle compositions targeted by an RGD peptideligand alone or in combination with Avastin treatment. Strongeranti-tumor efficacy has been observed using a combination cancertherapeutics with VEGFR2-siRNA inhibitor and Avastin (Bevacizumab) inthe same treatment regimen, in a human colon carcinoma (DLD-1) xenografttumor model.

FIG. 2. VEGFR2-siRNA nanoparticle targeting neovasculature throughsystemic delivery demonstrated potent efficacy in colon carcinomaxenograft mouse model (DLD-1 tumor) after four repeated administrationsevery three days. RT-PCR results illustrate the knockdown of VEGFR2 geneexpression at mRNA level.

FIG. 3. Human VEGFR2-siRNA nanoparticle targeting neovasculature throughsystemic delivery demonstrated potent efficacy in colon carcinomaxenograft mouse model (DLD-1 tumor) after four repeated administrationsevery three days. Immunohistochemistry images show down regulation ofVEGFR2 and CD31 expression in the tumor tissue.

FIG. 4. While human VEGFR2-siRNA nanoparticle targeting neovasculaturethrough systemic delivery demonstrated potent efficacy in coloncarcinoma xenograft mouse model (DLD-1 tumor), no significant IFN-αinduction was observed in the mouse blood stream 24 hours after the 2nddelivery.

FIG. 5. Human VEGF-siRNA (19 base pair with 3′ end overhangs) iseffective for silencing hVEGF expression in vitro and in vivo, at bothmRNA level and protein level, evaluated by RT-PCR and ELISA. Two upperfigures demonstrated significant hVEGF knockdown at both mRNA andprotein levels. The lower panel shows the anti-tumor activity ofsiRNA-mediated hVEGF knockdown in a MCF-7/VEGF165 cell induced xenograftmodel, where the hVEGF expression was significantly down regulated inthe tumor tissue.

FIG. 6. Human VEGF-siRNA (19 base pair with 3′ end overhangs) iseffective for silencing hVEGF expression in vivo resulting in tumorgrowth inhibition (HNSCC 1483 cell). Five times repeated intratumoraldelivery with electroporation enhancement with 7 day interval.

FIG. 7. Human VEGF-siRNA (25 basepair blunt end) is more effective forsilencing hVEGF expression in vitro and stronger than the 19 base pairsiRNA.

FIG. 8. Human VEGF-siRNA (25 basepair blunt end) is effective forsilencing hVEGF expression in vitro and has longer duration than that ofthe 19 basepair siRNA.

FIG. 9. Human VEGFR1-siRNA (25 base pair blunt end) is effective forsilencing hVEGFR1 expression in vitro after 48 hr.

FIG. 10. Human VEGFR1-siRNA (25 base pair blunt end) is effective forsilencing hVEGFR1 expression in vitro after 72 hr.

FIG. 11. Human VEGFR2-siRNA (25 base pair blunt end) is effective forsilencing hVEGFR2 expression in vitro at two different time points.

FIG. 12. Human VEGF-siRNA (25 base pair blunt end) is effective forsilencing hVEGF expression in vivo resulting in tumor growth inhibition,with a MDA-MB-435 breast carcinoma cell line.

FIG. 13. Concept framework for using siRNA cocktail targeting VEGF,VEGFR1 and VEGFR2 in the same treatment to block the angiogenesispathway.

FIG. 14. siRNA cocktail targeting VEGF, VEGFR1 and VEGFR2 is moreeffective than any single siRNA targeting individual gene at the samedosage tested in the HSV induced ocular angiogenesis mouse model, witheither local (left) or systemic (right) deliveries.

FIG. 15. Inhibition of angiogenesis by administration of VEGF pathwaysiRNA cocktail on day 17 or day 14 after induction of disease in oculartissues of a murine model of retinal angiogenesis in aperfusion/flatmount measurement.

FIG. 16. siRNA cocktail targeting VEGF, VEGFR1 and VEGFR2 demonstratesvery effective anti-angiogenesis activity in the retinopathy ofprematural (ROP) mouse model induced by Hypoxia.

FIG. 17. siRNA cocktail targeting VEGF₅ VEGFR1 and VEGFR2 demonstratesvery effective anti-angiogenesis activity in the retinopathy ofprematural (ROP) mouse model induced by Hypoxia.

FIG. 18. siRNA cocktail targeting VEGF, VEGFR1 and VEGFR2 inhibitsocular angiogenesis significantly with different routes ofadministration with different carriers. IP delivery was mediated by aligand-directed nanoparticle. siRNA Cocktail Mediated Knockdown of VEGFPathway Factors RT-PCR Detection of mRNA levels with ROP Model.

FIG. 19. Self-assembled nanoparticle for siRNA delivery. When thepre-made RGD-PEG-PEI conjugate aqueous solution is mixed with the siRNAaqueous solution, the nanoparticles will be self-assembled as described.The nanoparticles are relatively homogeneous in size, from 50 nm to 100nm.

FIG. 20. siRNA nanoparticle targeting neovasculature demonstrated tumortargeting property using labeled siRNA payload (left) and plasmidpayload expressing Luciferase reporter gene (right).

FIG. 21. VEGFR2-siRNA nanoparticle targeting neovasculature demonstratedpotent efficacy in neuroblastoma syngenic mouse model (N2A tumor) afterfour repeated administrations every three days.

FIG. 22. VEGFR2-siRNA nanoparticle targeting neovasculature demonstratedpotent efficacy in renal carcinoma xenograft mouse model (786-O tumor)after five repeated administrations every three days.

FIG. 23. VEGFR2-siRNA nanoparticle targeting neovasculature demonstratedpotent efficacy in colon carcinoma xenograft mouse model (DLD-1 tumor)after four repeated administrations every three days. Three differentdosages were used in the study.

FIG. 24. VEGFR2-siRNA nanoparticle targeting neovasculature demonstratedpotent efficacy in colon carcinoma xenograft mouse model (DLD-1 tumor)after four repeated administrations every three days. Four mg per kiloof VEGFR2 dosing resulted in the same anti-tumor efficacy as 5 mg perkilogram of Avastin.

FIG. 25. Manufacturing flow chart for production of an RGD-polymerconjugate and a nanoparticle-siRNA preparation starting from rawmaterials of a homobifunctional activated PEG, an RGD-2C peptide, apolyamine containing agent, and a mixture of dsRNA.

FIG. 26. The use of a static mixer to manufacture a nanoparticle-siRNApreparation starting from two solutions, a polymer solution comprisingpolymer conjugates and an siRNA solution comprising three dsRNA agents.

FIG. 27. 25-mer blunted double-stranded siRNA mediated an elongatedtarget gene silencing. MCF7/165 cells over-expressing hVEGF weretransfected with either regular 21-mer siRNA (19 basepair siRNA with 3′overhangs) (hVEGF-siRNA-a, FIG. 27 A, B, C) or 25-mer siRNA targetinghVEGF (hVEGF-25-siRNA-a, FIG. 27A; hVEGF-25-siRNA-b, FIG. 27B; andhVEGF-25-siRNA-C, FIG. 27C). The culture media from the transfectedcells were harvested at indicated time points post siRNA transfectionand the VEGF protein levels were measured using a commercial hVEGF ELISAkit. Data were presented as Mean+/−SD.

DETAILED DESCRIPTION

The present invention provides compositions and methods for treatment ofNV diseases, which typically are characterized by attributes of multipleproteins and abnormally over-expressed disease-causing genes andmultiple malfunctions of disease-causing proteins. The present inventionprovides nucleic acid agents, such as siRNA oligonucleotides, thatactivate RNA interference (RNAi) and are highly selective inhibitors ofgene expression with a sequence specific manner. The present inventionprovides inhibition of NV by modulation of protein activity includingreduction of protein expression levels and post transcriptionalmodification of proteins.

In cancer, the tumorigenesis process is thought to be the result ofabnormal over-expression of oncogenes, angiogenesis factors, growthfactors, and mutant tumor suppressors, even though under-expression ofother proteins also plays a critical role. Increasing evidences supportsthe notion that siRNA molecules are able to “knockdown” tumorigenicgenes both in vitro and in vivo, resulting in significant antitumoreffects. The compositions and methods of the present inventiondemonstrate substantial knockdown of human VEGF in MCF-7 cells,MDA-MB-435 cells and 1483 cell-induced xenograft tumor models, achievingtumor growth inhibition of 40-80%, using intratumoral delivery of siRNAspecifically targeting human VEGF pathway gene sequences. It isappreciated that inhibition of VEGF pathway gene expression inducesanti-angiogenesis effects that alter the microvasculature in tumors andthat activate tumor cell apoptosis and can enhance efficacy of cytotoxicchemotherapeutic drugs. However, to achieve significantly improvedantitumor efficacy of anti-angiogenesis agents and chemotherapeuticdrugs, a highly effective delivery method is necessary so that elevatedconcentrations of the drugs accumulate in the local tumor tissue, and inmany instances through a systemic administration.

The present inventors have described a method of validating drug targetsthat determines which targets control disease pathways and thereforejustify drug development efforts (see PCT/US02/31554). The presentinventors also have described technologies suitable for delivery ofnucleic acids into animal tissues. See WO01/47496, the contents of whichare hereby incorporated by reference in their entirety. These methodsenable administration of nucleic acid and achieve a significant (forexample, seven-fold) increase in efficiency compared to “gold standard”nucleotide delivery reagents. Accordingly, the methods provide strongactivity of nucleic acids in tissues including activity of candidatetarget proteins. This platform is a powerful tool for validation ofcandidate genes in a tissue.

In addition, the present inventors have used these methods to achievegene silencing in animal tissues, which is highly desired for validationof candidate target genes and as a therapeutic modality. Recently,double stranded RNA has been demonstrated to induce gene-specificsilencing by a phenomenon called RNA interference (RNAi). Although themechanism of RNAi still is not completely understood, early resultssuggested that the RNAi effect may be achieved in vitro in various celltypes including mammalian cells. A double stranded RNA targeted againsta target mRNA results in the degradation of the target, thereby causingthe silencing of the corresponding gene. Large double stranded RNA iscleaved into smaller fragments of 21-23 nucleotides long by an RNase IIIlike activity involving an enzyme Dicer. These shorter fragments, knownas siRNA (small interfering RNA), are believed to mediate the cleavageof mRNA.

Although gene down-regulation by the RNAi mechanism has been studied inC. elegans and other lower organisms in recent past, its effectivenessin mammalian cells in culture has only recently been demonstrated. AnRNAi effect recently was demonstrated in mouse using the fireflyluciferase gene reporter system. To develop an RNAi technology platformfor in vivo gene inhibition for research and for clinical application ofnucleic acid therapeutics to treat human diseases, the present inventorsperformed several in vivo studies in mouse models of disease. In thoseexperiments, either siRNA or dsRNA targeting a tumor related ligand(human VEGF) or receptor (mouse VEGFR2) was delivered to nude micebearing xenografted human MCF-7 derived tumor or human MDA-MB-435tumors. For the first time we were able to demonstrate that RNAi caneffectively silencing target gene in tumor cells in vivo and that, as aresult, tumor growth was inhibited.

The present inventors have achieved for the first time therapeuticcompositions and methods for treatment of a wide variety of NV diseasesusing dsRNA oligonucleotides inhibiting VEGF pathway genes. Theinvention is described here in detail, but one skilled in the art willappreciate the full extent of the invention.

A. Potent siRNA for VEGF Pathway Gene Inhibition

The present invention provides nucleic acid agents targeting andinhibiting VEGF pathway gene sequences with a variety of physicochemicalstructures. One preferred embodiment of the present invention usesnucleic acid agents called siRNA including 19 base pair dsRNA with 3′overhangs and 25 base pair dsRNA with blunt ends. The siRNA of theinvention with 25 base pair dsRNA with blunt ends were found to be someof the most potent inhibitors with some of the greatest duration ofinhibition. Additionally, incorporation of non-naturally occurringchemical analogues are useful in the invention, including 2′-O-Methylribose analogues of RNA, DNA and RNA chimeric oligonucleotides, andother chemical analogues of nucleic acid oligonucleotides. The 25basepair siRNA with and without 2′-O-Methyl ribose in the sense strandof the siRNA and targeting human VEGF sequence provide strong anddurable inhibitory effect, up to 70-80% in MCF-7/VEGF165 cells and intumors growing in animals. This inhibitory effect in MCF-7/VEGF165 cellculture lasted for 5 days. One aspect provided for by the invention issiRNA targeting human genes and the encoded sequence also targets othermammalian species such as other primates, mice, and rats but not limitedto these species. Methods well known to one skilled in the art can beused to identify and select siRNA provided by the invention. Many otherforms of siRNA targeting VEGF pathway genes are provided for by theinvention and others will be understood by one skilled in the art.

a. Human VEGF specific siRNA:

25 base pair blunt ends:

hVEGF-25-siRNA-a: Sense strand: 5′-r(CCUGAUGAGAUCGAGUACAUCUUCA)-3′(SEQ ID NO: 1) Antisense strand: 5′-r(UGAAGAUGUACUCGAUCUCAUCAGG)-3′.(SEQ ID NO: 2) hVEGF-25-siRNA-b: Sense strand:5′-r(GAGAGAUGAGCUUCCUACAGCACAA)-3′ (SEQ ID NO: 3) Antisense strand:5′-r(UUGUGCUGUAGGAAGCUCAUCUCUC)-3′. (SEQ ID NO: 4) hVEGF-25-siRNA-c:Sense strand: 5′-r(CACAACAAAUGUGAAUGCAGACCAA)-3′ (SEQ ID NO: 5)Antisense strand: 5′-r(UUGGUCUGCAUUCACAUUUGUUGUG)-3′ (SEQ ID NO: 6)

19 base pairs with two nucleotide (TT) overhangs at 3′:

(SEQ ID NO: 7) hVEGF165 5′-r (UCGAGACCCUGGUGGACAUTT)-3′b. Human VEGF receptor 1 specific siRNA:

25 base pair blunt ends:

hVEGFR1-25-siRNA-a, Sense strand: (SEQ ID NO: 8)5′-r(GCCAACAUAUUCUACAGUGUUCUUA)-3′ Antisense strand: (SEQ ID NO: 9)5′-r(UAAGAACACUGUAGAAUAUGUUGGC)-3′ hVEGFR1-25-siRNA-b, Sense strand:(SEQ ID NO: 10) 5′-r(CCCUCGCCGGAAGUUGUAUGGUUAA)-3′ Antisense strand:(SEQ ID NO: 11) 5′-r(UUAACCAUACAACUUCCGGCGAGGG)-3′.

19 basepairs with 2 3′ (TT) nucleotide overhangs:

(SEQ ID NO: 12) VEGF R1 (FLT) 5′-GGAGAGGACCUGAAACUGUTTc. Human VEGF receptor 2 specific siRNA:

25 basepair blunt ends:

hVEGFR2-25-siRNA-a, Sense strand: (SEQ ID NO: 13)5′-r(CCUCUUCUGUAAGACACUCACAAUU)-3′ Antisense strand: (SEQ ID NO: 14)5′-r(AAUUGUGAGUGUCUUACAGAAGAGG)-3′. hVEGFR2-25-siRNA-b, Sense strand:(SEQ ID NO: 15) 5′-r(CCCUUGAGUCCAAUCACACAAUUAA)-3′ Antisense strand:(SEQ ID NO: 16) 5′-r(UUAAUUGUGUGAUUGGACUCAAGGG)-3′. hVEGFR2-25--siRNA-c,Sense strand: (SEQ ID NO: 17) 5′-r(CCAAGUGAUUGAAGCAGAUGCCUUU)-3′Antisense strand: (SEQ ID NO: 18) 5′-r(AAAGGCAUCUGCUUCAAUCACUUGG)-3′.

19 basepairs with 2 3′ (TT) nucleotide overhangs:

(SEQ ID NO: 19) hVEGF R2 (KDR) 5′-CAGUAAGCGAAAGAGCCGGTT-3′

In another embodiment, the siRNA targeting human VEGF receptor 1 has thefollowing sequence:

Sense strand: (SEQ ID NO. 20) 5′-r(CCUCAAGAGCAAACGUGACUUAUUU)-3′Antisense strand: (SEQ ID NO. 21) 5′-r(AAAUAAGUCACGUUUGCUCUUGAGG)-3′.25 Base Pair siRNA Oligos can Target the Corresponding Genes from BothHuman and Mouse.

In another embodiment, the 25 base pair siRNA sequences were selected toagainst the corresponding mRNA sequences, including human VEGF, VEGFR1,VEGFR2, PDGFR-alpha, PDGFR-beta and EGFR genes. The sequences selectedare not only specific to the human genes but also specific to thecorresponding mouse genes, such as the sequences against human VEGFmRNAs are also against mouse VEGF mRNAs.

The 25 base pair siRNA oligos are very useful for mouse xenograft tumorstudy since the anti-tumor efficacy relies on knockdown both human andmouse genes in the tumor tissue. For example, knockdown VEGF expressionsfrom both tumor cells (human origin) and endothelium cells (mouseorigin) with the siRNA oligos targeting both sequences will providebetter anti-tumor efficacy.

Meanwhile, the toxicity study with the siRNA oligos targeting both genesfrom testing animals (e.g. mouse or monkey) and human genes will be veryuseful since the actual drugs were tested for both drug agent toxicologyand exaggerated pharmacology.

The following sequences are the 25 base pair siRNA oligo sequencestargeting VEGF, VEGFR2, VEGFR1, PDGFR-alpha, PDGFR-beta and EGFR mRNAsfrom both human and mouse genes, some of them can also target to theirmonkey and dog counterparts.

25 Base Pair VEGF siRNA Targeting Human, Mouse, Rat, Macaque, Dog VEGFmRNA Sequences:

mhVEGF25-1: sense, 5′-CAAGAUCCGCAGACGUGUAAAUGUU-3′; (SEQ ID NO. 22)antisense, 5′-AAC AUUUACACGUCUGCGGAUCUUG-3′ (SEQ ID NO. 23) mhVEGF25-2:sense, 5′-GCAGCUUGAGUUAAACGAACGUACU-3′; (SEQ ID NO. 24) antisense,5′-AGUACGUUCGUUUAACUCAAGCUGC-3′ (SEQ ID NO. 25) mhVEGF25-3: sense,5′-CAGCUUGAGUUAAACGAACGUACUU-3′; (SEQ ID NO. 26) antisense,5′-AAGUACGUUCGUUUAACUCAAGCUG-3′ (SEQ ID NO. 27) mhVEGF25-4: sense,5′-CCAUGCCAAGUGGUCCCAGGCUGCA-3′; (SEQ ID NO. 28) antisense,5′-TGCAGCCTGGGACCACTTGGCATGG-3′ (SEQ ID NO. 29) mhVEGF25-4: sense,5′-CACAUAGGAGAGAUGAGCUUCCUCA-3′; (SEQ ID NO. 30) antisense,5′-UGAGGAAGCUCAUCUCUCCUAUGUG-3′ (SEQ ID NO. 31)25 Base Pair VEGF R2 siRNA Sequences Targeting Both Human and MouseVEGFR2 mRNA Sequences:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 32)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 33) mhVEGFR225-2:sense, 5′-CUCAUGUCUGUUCUCAAGAUCCUCA-3′; (SEQ ID NO. 34) antisense:5′-UGAGGAUCUUGAGAACAGACAUGAG-3′ (SEQ ID NO. 35) mhVEGFR225-3: sense,5′-CUCAUGGUGAUUGUGGAAUUCUGCA-3′; (SEQ ID NO. 36) antisense:5′-UGCAGAAUUCCACAAUCACCAUGAG-3′ (SEQ ID NO. 37) mhVEGFR225-4: sense,5′-GAGCAUGGAAGAGGAUUCUGGACUC-3′; (SEQ ID NO. 38) antisense:5′-GAGUCCAGAAUCCTCUUCCAUGCTC-3′ (SEQ ID NO. 39) mhVEGFR225-5: sense,5′-CAGAACAGUAAGCGAAAGAGCCGGC-3′; (SEQ ID NO. 40) antisense:5′-GCCGGCUCUUUCGCUUACUGUUCUG-3′ (SEQ ID NO. 41) mhVEGFR225-6: sense,5′-GACUUCCUGACCUUGGAGCAUCUCA-3′; (SEQ ID NO. 42) antisense:5′-UGAGAUGCUCCAAGGUCAGGAAGUC-3′ (SEQ ID NO. 43) mhVEGFR225-7: sense,5′-CCUGACCUUGGAGCAUCUCAUCUGU-3′; (SEQ ID NO. 44) antisense:5′-ACAGAUGAGAUGCUCCAAGGUCAGG-3′ (SEQ ID NO. 45) mhVEGFR225-8: sense,5′-GCUAAGGGCAUGGAGUUCUUGGCAU-3′; (SEQ ID NO. 46) antisense:5′-AUGCCAAGAACUCCAUGCCCUUAGC-3′ (SEQ ID NO. 47)25 Base Pairs VEGF R1 siRNA Sequences Targeting Both Human and MouseVEGFR1 mRNA Sequences:

mhVEGFR125-1: sense, 5′-CACGCUGUUUAUUGAAAGAGUCACA-3′; (SEQ ID NO. 48)antisense: 5′-UGUGACUCUUUCAAUAAACAGCGUG-3′ (SEQ ID NO. 49) mhVEGFR125-2:sense, 5′-CGCUGUUUAUUGAAAGAGUCACAGA-3′; (SEQ ID NO. 50) antisense:5′-UCUGUGACUCUUUCAAUAAACAGCG-3′ (SEQ ID NO. 51) mhVEGFR125-3: sense,5′-CAAGGAGGGCCUCUGAUGGUGAUGU-3′; (SEQ ID NO. 52) antisense:5′-ACAUCACCAUCAGAGGCCCUCCUUG-3′ (SEQ ID NO. 53) mhVEGFR125-4: sense,5′-CCAACUACCUCAAGAGCAAACGUGA-3′; (SEQ ID NO. 54) antisense:5′-UCACGUUUGCUCUUGAGGUAGUUGG-3′ (SEQ ID NO. 55) mhVEGFR125-5: sense,5′-CUACCUCAAGAGCAAACGUGACUUA-3′; (SEQ ID NO. 56) antisense:5′-UAAGUCACGUUUGCUCUUGAGGUAG-3′ (SEQ ID NO. 57) mhVEGFR125-6: sense,5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 58) antisense:5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 59) mhVEGFR125-7: sense,5′-CAUUCAUCGGGACCUGGCAGCGAGA-3′; (SEQ ID NO. 60) antisense:5′-UCUCGCUGCCAGGUCCCGAUGAAUG-3′ (SEQ ID NO. 61) mhVEGFR125-8: sense,5′-CAUCGGGACCUGGCAGCGAGAAACA-3′; (SEQ ID NO. 62) antisense:5′-UGUUUCUCGCUGCCAGGUCCCGAUG-3′ (SEQ ID NO. 63) mhVEGFR125-9: sense,5′-GAGCCUGGAAAGAAUCAAAACCUUU-3′; (SEQ ID NO. 64) antisense:5′-AAAGGUUUUGAUUCUUUCCAGGCUC-3′ (SEQ ID NO. 65) mhVEGFR125-10: sense,5′-GCCUGGAAAGAAUCAAAACCUUUGA-3′; (SEQ ID NO. 66) antisense:5′-UCAAAGGUUUUGAUUCUUUCCAGGC-3′ (SEQ ID NO. 67) mhVEGFR125-11: sense,5′-GCCUGGAAAGAAUCAAAACCUUUGA-3′; (SEQ ID NO. 68) antisense:5′-UCAAAGGUUUUGAUUCUUUCCAGGC-3′ (SEQ ID NO. 69) mhVEGFR125-12: sense,5′-CUGAACUGAGUUUAAAAGGCACCCA-3′; (SEQ ID NO. 70) antisense:5′-UGGGUGCCUUUUAAACUGAGUUCAG-3′ (SEQ ID NO. 71) mhVEGFR125-13: sense,5′-GAACUGAGUUUAAAAGGCACCCAGC-3′; (SEQ ID NO. 72) antisense:5′-GCUGGGUGCCUUUUAAACUCAGUUG-3′ (SEQ ID NO. 73)25 Base Pairs siRNA Sequences Targeting Both Human and MousePDGFR-Alpha:

mhPDGFRa25-1: sense, 5′-GAAGAUAAUGACUCACCUGGGGCCA-3′; (SEQ ID NO. 74)antisense: 5′-UGGCCCCAGGUGAGUCAUUAUCUUC-3′ (SEQ ID NO. 75) mhPDGFRa25-2:sense, 5′-GAUAAUGACUCACCUGGGGCCACAU-3′; (SEQ ID NO. 76) antisense:5′-AUGUGGCCCCAGGUGAGUCAUUAUC-3′ (SEQ ID NO. 77) mhPDGFRa25-3: sense,5′-CUCACCUGGGGCCACAUUUGAACAU-3′; (SEQ ID NO. 78) antisense:5′-AUGUUCAAAUGUGGCCCCAGGUGAG-3′ (SEQ ID NO. 79) mhPDGFRa25-4: sense,5′-CUUGCUGGGAGCCUGCACCAAGUCA-3′; (SEQ ID NO. 80) antisense:5′-UGACUUGGUGCAGGCUCCCAGCAAG-3′ (SEQ ID NO. 81) mhPDGFRa25-5: sense,5′-GAUUCUACUUUCUACAAUAAGAUCA-3′; (SEQ ID NO. 82) antisense:5′-UGAUCUUAUUGUAGAAAGUAGAAUC-3′ (SEQ ID NO. 83) mhPDGFRa25-6: sense,5′-CAGAGACUGAGCGCUGACAGUGGCU-3′; (SEQ ID NO. 84) antisense:5′-AGCCACUGUCUGCGCUCAGUCUCUG-3′ (SEQ ID NO. 85) mhPDGFRa25-7: sense,5′-GACCUGGGCAAGAGGAACAGACACA-3′; (SEQ ID NO. 86) antisense:5′-UGUGUCUGUUCCUCUUGCCCAGGUC-3′ (SEQ ID NO. 87) mhPDGFRa25-8: sense,5′-CCACCUUCAUCAAGAGAGAGGACGA-3′; (SEQ ID NO. 88) antisense:5′-UCGUCCUCUCUCUUGAUGAAGGUGG-3′ (SEQ ID NO. 89) mhPDGFRa25-9: sense,5′-UAUGGAUUAAGCCGGUCCCAACCUGU-3′; (SEQ ID NO. 90) antisense:5′-ACAGGUUGGGACCGGCUUAAUCCAUA-3′ (SEQ ID NO. 91)25 Base Pairs siRNA Sequences Targeting Both Human and Mouse PDGFRbeta:

mhPDGFRB25-1: sense, 5′-CUGCAGAGACCUCAAAAGGUGUCCA-3′; (SEQ ID NO. 92)antisense: 5′-UGGACACCUUUUGAGGUCUCUGCAG-3′ (SEQ ID NO. 93) mhPDGFRB25-2:sense, 5′-CAGAGACCUCAAAAGGUGUCCACGU-3′; (SEQ ID NO. 94) antisense:5′-ACGUGGACACCUUUUGAGGUCUCUG-3′ (SEQ ID NO. 95) mhPDGFRB25-3: sense,5′-GUGGUGGUGAUCUCAGCCAUCCUGG-3′; (SEQ ID NO. 96) antisense:5′-CCAGGAUGGCUGAGAUCACCACCAC-3′ (SEQ ID NO. 97) mhPDGFRB25-4: sense,5′-GGUGGUGAUCUCAGCCAUCCUGGCC-3′; (SEQ ID NO. 98) antisense:5′-GGCCAGGAUGGCUGAGAUCACCACC-3′ (SEQ ID NO. 99) mhPDGFRB25-5:  sense,5′-GGCAAGCUGGUCAAGAUCUGUGACU-3′; (SEQ ID NO. 100) antisense:5′-AGUCACAGAUCUUGACCAGCUUGCC-3′ (SEQ ID NO. 101) mhPDGFRB25-6: sense,5′-GCAAGCUGGUCAAGAUCUGUGACUU-3′; (SEQ ID NO. 102) antisense:5′-AAGUCACAGAUCUUGACCAGCUUGC-3′ (SEQ ID NO. 103) mhPDGFRB25-7: sense,5′-GGGUGGCACCCCUUACCCAGAGCUG-3′; (SEQ ID NO. 104) antisense:5′-CAGCUCUGGGUAAGGGGUGCCACCC-3′ (SEQ ID NO. 105) mhPDGFRB25-8: sense,5′-GCACCCCUUACCCAGAGCUGCCCAU-3′; (SEQ ID NO. 106) antisense:5′-AUGGGCAGCUCUGGGUAAGGGGUGC-3′ (SEQ ID NO. 107) mhPDGFRB25-9: sense,5′-CUUACCCAGAGCUGCCCAUGAACGA-3′ (SEQ ID NO. 108) antisense:5′-UCGUUCAUGGGCAGCUCUGGGUAAG-3′ (SEQ ID NO. 109) mhPDGFRB25-10: sense,5′-CAUGCCUCCGACGAGAUCUAUGAGA-3′; (SEQ ID NO. 110) antisense:5′-UCUCAUAGAUCUCGUCGGAGGCAUG-3′ (SEQ ID NO. 111) mhPDGFRB25-11: sense,5′-GCCUCCGACGAGAUCUAUGAGAUCA-3′; (SEQ ID NO. 112) antisense:5′-UGAUCUCAUAGAUCUCGUCGGAGGC-3′ (SEQ ID NO. 113) mhPDGFRB25-12: sense,5′-CCGACGAGAUCUAUGAGAUCAUGCA-3′; (SEQ ID NO. 114) antisense:5′-UGCAUGAUCUCAUAGAUCUCGUCGG-3′ (SEQ ID NO. 115) mhPDGFRB25-13: sense,5′-GACGAGAUCUAUGAGAUCAUGCAGA-3′; (SEQ ID NO. 116) antisense:5′-UCUGCAUGAUCUCAUAGAUCUCGUC-3′ (SEQ ID NO. 117)25 Base Pairs siRNA Sequences Targeting Both Human and Mouse EGFR:

mhEGFR-1: sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 118)antisense: 5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 119) mhEGFR-2:sense, 5′-CAGCAUGUCAAGAUCACAGAUUUUG-3′; (SEQ ID NO. 120) antisense:5′-CAAAAUCUGUGAUCUUGACAUGCUG-3′ (SEQ ID NO. 121) mhEGFR-3: sense,5′-GAUCACAGAUUUUGGGCUGGCCAAA-3′; (SEQ ID NO. 122) antisense:5′-UUUGGCCAGCCCAAAAUCUGUGAUC-3′ (SEQ ID NO. 123) mhEGFR-4: sense,5′-CAGAUUUUGGGCUGGCCAAACUGCU-3′ (SEQ ID NO. 124) antisense:5′-AGCAGUUUGGCCAGCCCAAAAUCUG-3′ (SEQ ID NO. 125) mhEGFR-5: sense,5′-C AAAGUGCCUAUCAAGUGGAUGGCA-3′ (SEQ ID NO. 126) antisense:5′-UGCCAUCCACUUGAUAGGCACUUUG-3′ (SEQ ID NO. 127) mhEGFR-6: sense,5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′; (SEQ ID NO. 128) antisense:5′-ACCAUGAUCAUGUAGACAUCGAUGG-3′ (SEQ ID NO. 129) mhEGFR-7: sense,5′-CGAUGUCUACAUGAUCAUGGUCAAGU-3′; (SEQ ID NO. 130) antisense:5′-ACUUGACCAUGAUCAUGUAGACAUCG-3′ (SEQ ID NO. 131) mhEGFR-8: sense,5′-CUACAUGAUCAUGGUCAAGUGCUGG-3′; (SEQ ID NO. 132) antisense:5′-CCAGCACUUGACCAUGAUCAUGUAG-3′ (SEQ ID NO. 133) mhEGFR-9: sense,5′-CAUGAUCAUGGUCAAGUGCUGGAUGA-3′; (SEQ ID NO. 134) antisense:5′-UCAUCCAGCACUUGACCAUGAUCAUG-3′ (SEQ ID NO. 135) mhEGFR-10: sense,5′-GGAUGAAAGAAUGCAUUUGCCAAGU-3′; (SEQ ID NO. 136) antisense:5′-ACUUGGCAAAUGCAUUCUUUCAUCC-3′ (SEQ ID NO. 137) mhEGFR-11: sense,5′-GACAACCCUGACUACCAGCAGGACU-3′ (SEQ ID NO. 138) antisense:5′-AGUCCUGCUGGUAGUCAGGGUUGUC-3′ (SEQ ID NO. 139) mhEGFR-12: sense,5′-CCUUCUUAAAGACCAUCCAGGAGGU-3′; (SEQ ID NO. 140) antisense:5′-ACCUCCUGGAUGGUCUUUAAGAAGG-3′ (SEQ ID NO. 141)

Another embodiment is use of a combination of these oligos in the samedrug payload. When three or more of those 25 base pair siRNAs targetingthree or more genes involving in the disease process were packaged inthe same nanoparticles and delivered through the same administrationrouts (or different routes), the treatment is e more effective since theknockdown of multiple gene expressions can effectively block theparticular tumorigenic pathway. The following combinations can befurther expended using the same general principles applied here.

Combinations of 25 Base Pair siRNA Oligos, Each of them can Target theCorresponding Genes from Both Human and Mouse, can Achieve More PotentTherapeutic (Multi-targeted) Efficacy:

1. Targeting VEGF Pathways:

VEGF-VEGFR1-VEGFR2 cocktail A:

mhVEGF25-1: sense, 5′-CAAGAUCCGCAGACGUGUAAAUGUU-3′; (SEQ ID NO. 142)antisense, 5′-AACAUUUACACGUCUGCGGAUCUUG-3′ (SEQ ID NO. 143)mhVEGFR125-3: sense, 5′-CAAGGAGGGCCUCUGAUGGUGAUGU-3′; (SEQ ID NO. 144)antisense: 5′-ACAUCACCAUCAGAGGCCCUCCUUG-3′ (SEQ ID NO. 145)mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 146)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 147)VEGF-VEGFR1-VEGFR2 cocktail B:

mhVEGF25-1: sense, 5′-CAAGAUCCGCAGACGUGUAAAUGUU-3′; (SEQ ID NO. 148)antisense, 5′-AACAUUUACACGUCUGCGGAUCUUG-3′ (SEQ ID NO. 149)mhVEGFR125-6: sense, 5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 150)antisense: 5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 151)mhVEGFR225-2: sense, 5′-CUCAUGUCUGUUCUCAAGAUCCUCA-3′; (SEQ ID NO. 152)antisense: 5′-UGAGGAUCUUGAGAACAGACAUGAG-3′ (SEQ ID NO. 153)VEGF-VEGFR1-VEGFR2 cocktail C:

mhVEGF25-2: sense, 5′-GCAGCUUGAGUUAAACGAACGUACU-3′; (SEQ ID NO. 154)antisense, 5′-AGUACGUUCGUUUAACUCAAGCUGC-3′ (SEQ ID NO. 155)mhVEGFR125-6: sense, 5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 156)antisense: 5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 157)mhVEGFR225-2: sense, 5′-CUCAUGUCUGUUCUCAAGAUCCUCA-3′; (SEQ ID NO. 158)antisense: 5′-UGAGGAUCUUGAGAACAGACAUGAG-3′ (SEQ ID NO. 159)

Many other combinations with the 25 base pair siRNA oligos targetingVEGF, VEGFR1 and VEGFR2 respectively can be composed for the bestanti-angiogenesis efficacy, by either equal or different ratios of eachcomponents.

2. Targeting Both VEGF and EGF Pathways:

VEGF-VEGFR2-EGFR cocktail A:

mhVEGF25-1: sense, 5′-CAAGAUCCGCAGACGUGUAAAUGUU-3′; (SEQ ID NO. 160)antisense, 5′-AACAUUUACACGUCUGCGGAUCUUG-3′ (SEQ ID NO. 161)mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 162)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 163) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 164) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 165)VEGF-VEGFR2-EGFR cocktail B:

mhVEGF25-1: sense, 5′-CAAGAUCCGCAGACGUGUAAAUGUU-3′; (SEQ ID NO. 166)antisense, 5′-AACAUUUACACGUCUGCGGAUCUUG-3′ (SEQ ID NO. 167)mhVEGFR225-2: sense, 5′-CUCAUGUCUGUUCUCAAGAUCCUCA.3′; (SEQ ID NO. 168)antisense: 5′-UG AGGAUCUUGAGAACAGACAUGAG-3′ (SEQ ID NO. 169) mhEGFR-5:sense, 5′-CAAAGUGCCUAUCAAGUGGAUGGCA-3′; (SEQ ID NO. 170) antisense:5′-UGCCAUCCACUUGAUAGGCACUUUG-3′ (SEQ ID NO. 171)VEGF-VEGFR2-EGFR cocktail C:

mhVEGF25-2: sense, 5′-GCAGCUUGAGUUAAACGAACGUACU-3′; (SEQ ID NO. 172)antisense, 5′-AGUACGUUCGUUUAACUCAAGCUGC-3′ (SEQ ID NO. 173)mhVEGFR225-2: sense, 5′-CUCAUGUCUGUUCUCAAGAUCCUCA-3′; (SEQ ID NO. 174)antisense: 5′-UGAGGAUCUUGAGAACAGACAUGAG-3′ (SEQ ID NO. 175) mhEGFR-9:sense, 5′-CAUGAUCAUGGUCAAGUGCUGGAUGA-3′; (SEQ ID NO. 176) antisense:5′-UCAUCCAGCACUUGACCAUGAUCAUG-3′ (SEQ ID NO. 177)

Many other combinations can be made with other sequences targeting thosethree genes with different composition and equal or different rations ofthe siRNA oligos.

3. Targeting VEGF, PDGF and EGF Pathways:

VEGFR2-PDGFRa-EGFR cocktail A:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′ (SEQ ID NO. 178)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 179)mhPDGFRa25-1: sense, 5′-GAAGAUAAUGACUCACCUGGGGCCA-3′; (SEQ ID NO. 180)antisense: 5′-UGGCCCCAGGUGAGUCAUUAUCUUC-3′ (SEQ ID NO. 181) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 182) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 183)VEGFR1-VEGFR2-PDGFRa-EGFR cocktail B:

mhVEGFR125-6: sense, 5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 184)antisense: 5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 185)mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 186)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 187)mhPDGFRa25-2: sense, 5′-GAUAAUGACUCACCUGGGGCCACAU-3′; (SEQ ID NO. 188)antisense: 5′-AUGUGGCCCCAGGUGAGUCAUUAUC-3′ (SEQ ID NO. 189) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 190) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 191)VEGFR2-PDGFRa-EGFR cocktail C:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 192)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 193)mhPDGFRa25-2: sense, 5′-GAUAAUGACUCACCUGGGGCCACAU-3′; (SEQ ID NO. 194)antisense: 5′-AUGUGGCCCCAGGUGAGUCAUUAUC-3′ (SEQ ID NO. 195) mhEGFR-12:sense, 5′-CCUUCUUAAAGACCAUCCAGGAGGU-3′; (SEQ ID NO. 196) antisense:5′-ACCUCCUGGAUGGUCUUUAAGAAGG-3′ (SEQ ID NO. 197)VEGFR2-PDGFRb-EGFR cocktail A:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 198)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 199)mhPDGFRB25-5: sense, 5′-GGCAAGCUGGUCAAGAUCUGUGACU-3′; (SEQ ID NO. 200)antisense: 5′-AGUCACAGAUCUUGACCAGCUUGCC-3′ (SEQ ID NO. 201) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 202) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 203)VEGFR2-PDGFRb-EGFR cocktail B:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 204)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 205)mhPDGFRB25-10: sense, 5′-CAUGCCUCCGACGAGAUCUAUGAGA-3′; (SEQ ID NO. 206)antisense: 5′-UCUCAUAGAUCUCGUCGGAGGCAUG-3′ (SEQ ID NO. 207) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 208) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 209)VEGFR2-PDGFRb-EGFR cocktail C:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 210)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 211)mhPDGFRB25-5: sense, 5′-GGCAAGCUGGUCAAGAUCUGUGACU-3′; (SEQ ID NO. 212)antisense: 5′-AGUCACAGAUCUUGACCAGCUUGCC-3′ (SEQ ID NO. 213) mhEGFR-12:sense, 5′-CCUUCUUAAAGACCAUCCAGGAGGU-3′; (SEQ ID NO. 214) antisense:5′-ACCUCCUGGAUGGUCUUUAAGAAGG-3′ (SEQ ID NO. 215)

Many other combinations can be made with other sequences targeting thosethree genes with different composition and equal or different ratios ofthe siRNA oligos.

The combinations can also be made with more than three sequences:

VEGFR2-PDGFRab-EGFR cocktail A:

mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 216)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 217)mhPDGFRa25-2: sense, 5′-GAUAAUGACUCACCUGGGGCCACAU-3′ (SEQ ID NO. 218)antisense: 5′-AUGUGGCCCCAGGUGAGUCAUUAUC-3′ (SEQ ID NO. 219)mhPDGFRb25-5: sense, 5′-GGCAAGCUGGUCAAGAUCUGUGACU-3′; (SEQ ID NO. 220)antisense: 5′-AGUCACAGAUCUUGACCAGCUUGCC-3′ (SEQ ID NO. 221) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 222) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 223)VEGFR1-VEGFR2-PDGFRb-EGFR-VEGF cocktail A:

mhVEGFR125-6: sense, 5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 224)antisense: 5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 225)mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′; (SEQ ID NO. 226)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 227)mhPDGFRB25-10: sense, 5′-CAUGCCUCCGACGAGAUCUAUGAGA-3′; (SEQ ID NO. 228)antisense: 5′-UCUCAUAGAUCUCGUCGGAGGCAUG-3′ (SEQ ID NO. 229) mhEGFR-1:sense, 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′; (SEQ ID NO. 230) antisense:5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′ (SEQ ID NO. 231) mhVEGF25-2: sense,5′-GCAGCUUGAGUUAAACGAACGUACU-3′; (SEQ ID NO. 232) antisense:5′-AGUACGUUCGUUUAACUCAAGCUGC-3′ (SEQ ID NO. 233)VEGFR1-VEGFR2-PDGFRb-EGFR-VEGF cocktail C:

mhVEGFR125-6: sense, 5′-CCAGAAAGUGCAUUCAUCGGGACCU-3′; (SEQ ID NO. 234)antisense: 5′-AGGUCCCGAUGAAUGCACUUUCUGG-3′ (SEQ ID NO. 235)mhVEGFR225-1: sense, 5′-CCUACGGACCGUUAAGCGGGCCAAU-3′ (SEQ ID NO. 236)antisense: 5′-AUUGGCCCGCUUAACGGUCCGUAGG-3′ (SEQ ID NO. 237)mhPDGFRa25-2: sense, 5′-GAUAAUGACUCACCUGGGGCCACAU-3′ (SEQ ID NO. 238)antisense: 5′-AUGUGGCCCCAGGUGAGUCAUUAUC-3′ (SEQ ID NO. 239)mhPDGFRB25-5: sense, 5′-GGCAAGCUGGUCAAGAUCUGUGACU-3′; (SEQ ID NO. 240)antisense: 5′-AGUCACAGAUCUUGACCAGCUUGCC-3′ (SEQ ID NO. 241) mhEGFR-12:sense, 5′-CCUUCUUAAAGACCAUCCAGGAGGU-3′; (SEQ ID NO. 242) antisense:5′-ACCUCCUGGAUGGUCUUUAAGAAGG-3′ (SEQ ID NO. 243) mhVEGF25-2: sense,5′-GCAGCUUGAGUUAAACGAACGUACU-3′ (SEQ ID NO. 244) antisense,5′-AGUACGUUCGUUUAACUCAAGCUGC-3′ (SEQ ID NO. 245)

B. Combined VEGF Pathway Gene Inhibition

A number of factors were considered during development of thecompositions and methods of the present invention for inhibition of NV.First, NV diseases are complex and the result of multiple proteins andabnormally over-expressed disease-causing genes and multiplemalfunctions of disease-causing proteins. Second, nucleic acid agentsthat activate RNA interference (RNAi) are highly selective inhibitors ofgene expression in a sequence specific manner. Thirdly, inhibition of NVby modulation of protein activity can be achieved by a number ofmethods, including an inhibition of protein function (antagonists),stimulation of protein function (agonists), reduction of proteinexpression levels, and post transcriptional modification of proteins.Treatment of NV disease ideally requires sophisticated therapeuticactions to effectively shut down a particular biological pathway that iscritical for disease progression, including simultaneously blockingfunctions of both ligands and their receptor, simultaneously blockingreceptor activity and down stream signaling proteins, and simultaneouslyblocking redundant elements of a pathway. This is the case for the NVdisease and the VEGF pathway.

However, identification of a single agent selective for two or three ormore proteins is difficult and oftentimes impractical, if notimpossible. To overcome this difficulty, use of a combination of drugshas been something of a trend in modern medicine. In oncologyapplications, combined chemotherapies have achieved remarkableanti-cancer efficacy. One example is the use of docetaxel, ifosfamideand cisplatin combination therapy for treatment of oropharyngeal cancerwith multiple bone metastases from prostate cancer (2). In othertherapeutic areas, another example is the treatment of ulcerativecolitis with combination of Corticosteroids, Metronidazole andVancomycin (3). For treatment of insufficiently controlled type 2diabetes, the efficacy and safety of adding rosiglitazone to acombination of glimepiride and metformin therapy were evaluated (4).

Although those clinical studies have demonstrated remarkable therapeuticefficacies, the toxicities of higher dosage and long time safeties arealways major concerns, due to their different sources of origins,different manufacturing processes and different chemistry properties. Toovercome these problems, an aspect of the present invention are using ssiRNA oligonucleotide gene inhibitors to provide a unique advantage toachieve combination effects with a combination of siRNA to targetmultiple disease causing genes in the same treatment. One advantageprovided by the present invention is a result that all siRNAoligonucleotides are very similar chemically and pharmacologically, andcan be from the same source of origin and same manufacturing process.Another advantage provided by the present invention is that multiplesiRNA oligonucleotides can be formulated in a single preparation such asa nanoparticle preparation.

Accordingly, an aspect of the present invention is to combine siRNAagents so as to achieve specific and selective inhibition of multipleVEGF pathway genes and as a result achieve a inhibition of NV diseaseand a better clinical benefit. The present invention provides for manycombinations of siRNA targeting including combinations that target VEGFitself together with its receptors including VEGF R1 (Flt1) and VEGF R2(KDR), parallel growth factors including PDGF and EGF and theirreceptors, down stream signaling factors including RAF and AKT, andtranscription factors including NFKB, and their combination. A preferredembodiment is a combination of siRNA inhibiting VEGF and two of itsreceptors VEGF R1 (Flt1) and VEGF R2 (KDR). Another preferred embodimentis a combination of siRNA inhibiting VEGF and its receptors, PDGF andits receptors, and EGF and its receptors. Yet another preferredembodiment is a combination of siRNA inhibiting VEGF and its receptorsand down stream signaling.

The dsRNA oligonucleotides can be combined for a therapeutic for thetreatment of NV disease. In one embodiment of the present invention theycan be mixed together as a cocktail and in another embodiment they canbe administered sequentially by the same route or by different routesand formulations and in yet another embodiment some can be administeredas a cocktail and some administered sequentially. Other combinations ofsiRNA and methods for their combination will be understood by oneskilled in the art to achieve treatment of NV diseases.

C. Combined VEGF Pathway Antagonist and Gene Inhibition

Disease is complicated and often involves multiple pathologicalprocesses as well as variations in severity of disease symptoms and,often, variations from one patient to another. Many diseases are causedby abnormal overexpression of disease-causing or disease control genes,or from foreign infectious organisms, or both. Disease progression, thedevelopment of reduced response to treatments over time and drugresistance also limit clinical benefit of a single treatment ormodality. One means to overcome such limitations is through use ofcombinations of treatments and drugs.

Therefore, an aspect of the present invention is to combine siRNA agentswith other agents so as to achieve a strong, durable, and robustinhibition of NV disease and a superior clinical benefit. The presentinvention provides multiple combinations of siRNA agents together withother agents, including combinations of therapeutic siRNA for VEGFitself and its receptors with antagonists of VEGF itself and itsreceptors (such as Avastin). The present invention also providescombinations of therapeutic siRNA agents with orally available kinaseinhibitors (such as SU1 1248). The present invention also providescombinations of therapeutic siRNA agents with immunotherapy. Yet anotherembodiment of the present invention is to combine siRNA withantiproliferative agents. Other combinations of siRNA agents and otheragents will be understood by one skilled in the art to achieve treatmentof NV diseases.

D. Formulation and Administration

Recent efforts towards developing tissue targetable nucleic aciddelivery systems based on synthetic reagents have produced promisingresults. To be robust, effective delivery systems should have multiplelevels of selectivity, i.e. selective localization at the disease tissueand selective inhibition of biochemical pathways driving the pathology.Moreover, the most effective current therapies require “multi-targeted”therapeutics, i.e. designer “dirty” drugs with multiple mechanisms ofactivity, blocking redundant pathological pathways. A superior approachwould be use “smart” nanoparticles that simultaneously target diseaseand deliver nucleic acid agents into the target cells and into thecorrect subcellular compartment.

In one embodiment, the present invention provides for formulations forsiRNA dsRNA oligonucleotides that comprise tissue-targetable deliverywith three additional properties. These properties are nucleic acidbinding into a core that can release the siRNA into the cytoplasm,protection from non-specific interactions, and tissue targeting thatprovides cell uptake. No one material has all of these requiredproperties in one molecule. The invention provides for compositions andmethods that use modular conjugates of three materials to combine andassemble the multiple properties required. They can be designed andsynthesized to incorporate various properties and then mixed with thesiRNA payload to form the nanoparticles. Based on these embodiments, apreferred embodiment comprises a modular polymer conjugate targetingneovasculature by coupling a peptide ligand specific for those cells toone end of a protective polymer, coupled at its other end to a cationiccarrier for nucleic acids. This polymer conjugate has three functionaldomains, sometimes referred to as a tri-functional polymer (TFP). Themodular design of this conjugate allows replacement and optimization ofeach component separately. An alternative approach has been to attachsurface coatings onto preformed nanoparticles. Adsorption of a stericpolymer coating onto polymers is self-limiting; once a steric layerbegins to form it will impede further addition of polymer. Thecompositions and methods of the invention permit an efficient method foroptimization of each of the three functions, largely independent of theother two functions.

Formation of Nucleic Acid Core Particle

Delivery agents for nucleic acids must assist their gaining access tothe interior of cells and in a manner such that they can exert theirbiological activity. Efforts to address this challenge for nucleic acidtherapeutics with synthetic materials include use of simple cationiclipid and polymer complexes developed as in vitro DNA transfectionreagents. It was quickly found that both getting nucleic acids intocells and obtaining biological activity is extremely difficult. Forexample, achieving a stable nucleic acid package for transport ispossible but not always easy to reconcile with the need to release thenucleic acid into the nucleus, or in the case of siRNA into thecytoplasm. Also, a large number of cationic lipids and polymers that areeffective in vitro do not retain activity when administered in vivo,unfortunately for reasons still largely unknown, providing littlepredictive value for developing complexes active in vivo. Interest instudies with RNA have lagged far behind, until now with the recenteruption of interest in siRNA. Nonetheless, lung tissue can often betransfected from an intravenous administration of DNA complexes. Similarbiological activity has been observed when siRNA has been used as thepayload.

Recent studies have identified one class of cationic polymers composedof defined polypeptide structures that appear to have broadcapabilities. A large number of members of this class can be synthesizedwith defined structures covering linear and branched forms and have beenfound to offer biological activity both in vitro and in vivo. They havebeen shown to have activity with several types of nucleic acids,including plasmids and DNA or RNA oligonucleotides. The success withthis class of cationic polymer appears to result from a designincorporating specific structures, including branching, and using amixture of hard and weak bases to form a polymer with mixed cationicproperties. Another advantage of this particular class is itsbiodegradable nature, being constructed entirely from natural aminoacids, albeit non-natural branching. Having a polymer with at least twoproperties to use for optimization, continuing development of materialsfor forming effective core complexes with siRNA can be refined andfocused on for further improvements to balance stability and cytoplasmicrelease. Optimized core forming polymers can be used as the basis forthe other functions.

Protective Steric Coating

Even liposomes with an external lipid bilayer resembling the outercellular membrane are rapidly recognized and cleared from blood. Unlikeviral particles, though, nanotechnology has access to a broad range ofsynthetic polymer chemistry. Hydrophilic polymers, such as PEG andpolyacetals and polyoxazolines, have proven effective to form a “steric”protective layer on the surface of colloidal drug delivery systemswhether liposomes, polymer or electrostatic nanoparticles, reducingimmune clearance from blood. The use of this steric PEG layer was firstdeveloped and most extensively studied with sterically stabilizedliposomes. The present invention provides for alternative approaches,such as chemical reduction of surface charge, in addition to a stericpolymer coating.

The evidence is strong that the steric barrier, and biologicalconsequences, derive from physical, not chemical, properties. Severalother hydrophilic polymers have been reported as alternatives to PEG.Physical studies on sterically stabilized liposomes have provided astrong mechanistic underpinning for physical behavior of the polymerlayer and can be used to achieve similar coatings on other types ofparticles. However, while physical studies have shown formation of asimilar polymer layer on the surface of polymer complexes with nucleicacids, and achievement of similar biological properties, we lacksufficient information today to use of the physical properties toaccurately predict the desired biological properties, protection fromimmune clearance from blood.

Advanced studies are constructing nanoparticle complexes with controlledvariations in their physical properties followed by determination oftheir biological properties. From the many liposome studies, it is clearthan physical properties with the greatest impact on biological activitycan be obtained by synthesis of a matrix of conjugates varying size ofthe two polymers and the grafting density. Note that while the surfacesteric layer function is due to physical properties, the optimalconjugation chemistry still depends on the specific chemical nature ofthe steric polymer and the carrier to which it is coupled.

Methods for formation of the nanoparticles with the surface stericpolymer layer are also a relevant parameter. One embodiment of thepresent invention provides for methods where the steric polymer iscoupled to the carrier polymer to give a conjugate that self-assembleswith the nucleic acid forming a nanoparticle with the steric polymersurface layer. Another embodiment is to attach surface coatings ontopreformed nanoparticles. In self-assembly formation of the surfacesteric layer depends on interactions of the carrier polymer with thepayload, not on penetration through a forming steric layer to react withparticle surface. In this case, effects of the steric polymer on abilityof the carrier polymer to bind the nucleic acid payload may have adverseeffects on particle formation, and thus the surface steric layer. Ifthis occurs, the grafting density of the steric polymer on the carrierwill have exceeded its maximum, or the structural nature of the graftingis not adequate.

Surface Exposed Ligands Targeting Specific Tissues

Ability of the nanoparticle to reach the interior of the target cells,selectively, resides in its ability to induce a specific receptormediated uptake. This is provided in the present invention by exposedligands with the binding specificity. While many types of ligands havebeen studied extensively for targeting colloidal delivery systems, manyhave relied on antibodies. Over twenty years ago, this idea wasdeveloped for coupling antibodies to the surface of liposomes, usuallyreferred to as immunoliposomes. One important parameter that has emergedin the impact of ligand density. The extensive studies achieved goodsuccess in vitro but only recently have begun to produce positiveresults in vivo, now reaching the stage of clinical development fordelivery of small molecule drugs.

Antibodies tend to meet many requirements for use as the ligand,including good binding selectivity and nearly routine preparation fornearly any receptor and broad applicability of protein coupling methodsregardless of nanoparticle type. Monoclonal antibodies even show signsof utility to cross the blood-brain-barrier. On the other hand, they arenot ideal. As targeting ligands, antibodies are very large proteins,about 150 Kd. This makes conjugation to yield a specific orientationdifficult unless an extra Cys amino acid residue can be introduced, orother unique site identified, for the chemical coupling. Truncated formscan be used to reduce size but loss of affinity can occur, such as with“scFv” constructions.

Another troubling property of antibodies is their multitude ofbiological activities, many conveyed in domains not associated withantigen binding, which are part of their role in the mammalian immunesystem. These immune activities can be exploited but they also caninterfere in the nanoparticle steric layer role of avoiding immuneclearance. Other proteins that are natural ligands also have beenconsidered for targeting nanoparticles, such as transferrin fortransferrin receptor and Factor VII and Factor VIIa proteins and theirfragments. These agents can be good candidates but their successful userequires considerable effort to identify specific coupling chemistry,coupling without loss of binding activity, and exposure on the particlewithout reintroducing immune clearance.

A preferred class of ligands are small molecular weight compounds withstrong selective binding affinity for internalizing receptors. Studieshave been evaluated natural metabolites such as vitamins like folate andthiamine, polysaccharides like wheat germ agglutinin or sialyl Lewis^(x)for e-selectin, and peptide binding domains like RGD for integrins.Peptides offer a versatile class of ligand, since phage displaylibraries can be used to screen for natural or unnatural sequences, evenwith in vivo panning methods. Such phage display methods can permitretention of an unpaired Cys residue at one end for ease of couplingregardless of sequence. The recent in vivo success using an RGD peptidefor targeted delivery of nanoparticles to neovasculature indicates thisapproach can be very effective to meet the major requirements foreffective ligands: specific chemistry that doesn't interfere in ligandbinding or induce immune clearance yet enables selective receptormediated uptake at the target cells.

A preferred embodiment of the invention comprises an RGD-2C peptideligand conjugate. The RGD-2C ligand provides localization atneovasculature as well as tumor cells nearby tumor neovasculature,intracellular internalization of the nucleic acid containingnanoparticles, and release of the nucleic acid giving effectivebiological activity of the nucleic acid.

Another preferred embodiment of the invention comprises mixtures ofligand-conjugates of two or more ligands, or conjugates of two or moreligands. A preferred embodiment of the invention comprises mixtures ofRGD peptide ligands with Factor VII proteins or their peptide fragmentsor chemical analogues. Another preferred embodiment of the inventioncomprises mixtures of RGD peptide ligands with ligands bindinge-selectins. Another preferred embodiment of the invention comprisesmixtures of RGD peptide ligands with apoE proteins or their peptidefragments or chemical analogues. Another preferred embodiment of theinvention comprises mixtures of RGD peptide ligands with tumor cellbinding ligands such as folate, transferrin, porphyrin, bFGF, and EGF.Another preferred embodiment of the invention comprises mixtures ofthree or more of these ligands. A preferred embodiment of the inventioncomprises a conjugate of two or more ligands at one domain of a carrierand a polyamine agent at a second domain of said carrier.

Another preferred embodiment of the invention comprises mixtures ofligand conjugates. The mixture of ligand conjugates can comprise theligands conjugated to the same carrier agent and optionally to the samedomain of the carrier or the mixture of ligand conjugates can comprisethe ligands conjugated to different carrier agents or optionally todifferent domains of carrier agents. The mixture of ligand conjugatescan be further comprised of protective, fusogenic, or carrier agents,such as PEG or polyacetals or pH-sensitive polymers or polyamines orhistidine-lysine copolymers.

The compositions and methods of the present invention provide foradministration of RNAi comprising polymers, polymer conjugates, lipids,micelles, self-assembly colloids, nanoparticles, sterically stabilizednanoparticles, or ligand-directed nanoparticles. Targeted syntheticvectors of the type described in WO01/49324, which is herebyincorporated by reference in its entirety, may be used for systemicdelivery of nucleic acids of the present invention inducing RNAi. In oneembodiment, a PEI-PEG-RGD (polyethyleneimine-polyethyleneglycol-argine-glycine-aspartic acid) synthetic vector can be preparedand used, for example as in Examples 53 and 56 of WO01/49324. Thisvector was used to deliver RNAi systemically via intravenous injection.The skilled artisan will recognize that other targeted synthetic vectormolecules known in the art may be used. For example, the vector may havean inner shell made up of a core complex comprising the RNAi and atleast one complex forming reagent.

The vector also may contain a fusogenic moiety, which may comprise ashell that is anchored to the core complex, or may be incorporateddirectly into the core complex. The vector may further have an outershell moiety that stabilizes the vector and reduces nonspecific bindingto proteins and cells. The outer shell moiety may comprise a hydrophilicpolymer, and/or may be anchored to the fusogenic moiety. The outer shellmoiety may be anchored to the core complex. The vector may contain atargeting moiety that enhances binding of the vector to a target tissueand cell population. Suitable targeting moieties are known in the artand are described in detail in WO01/49324.

a. Preparation of RGD-Targeted Nanoparticles Containing VEGF PathwaysiRNA:

One embodiment of the present invention provides compositions andmethods for RGD-mediated ligand-directed nanoparticle preparations ofanti-VEGF pathway siRNA short dsRNA molecules. FIGS. 25 and 26 show amethod for the manufacture of RGD-2C-mediated tissue targetednanoparticle containing siRNA. The targeting ligand, RGD containingpeptide (ACRGDMFGCA) (SEQ ID NO: 246) is conjugated to a steric polymersuch as polyethylene glycol, or other polymers with similar properties.This ligand-steric polymer conjugate is further conjugated to apolycation such as polyethyleneimine or other effective material such asa histidine-lysine copolymer. The conjugates can be covalent ornon-covalent bonds and the covalent bonds can be non-cleavable or theycan be cleavable such as by hydrolysis or by reducing agents. Theconjugates can be prepared with hetero-bifunctional polymers, such as anNHS-PEG-VS commercially available reagent (Nektar) orhetero-bifunctional agents prepared for manufacturing, or withhomo-bifunctional polymers, such as NHS-PEG-NHS commercially availablereagent (Rapp Polymere) or homo-bifunctional agents prepared formanufacturing. One preferred embodiment of the present inventioncomprises use of a homo-bifunctional NHS-PEG-NHS agent, such as areagent commercially available from RAPP Polymere, first to couple withan RGD-2C peptide prepared by solid phase synthesis and send to couplewith a polyamine containing polymer such as PEI commercially availableor a histidine-lysine copolymer prepared by solid phase synthesis.

Another preferred embodiment of the present invention comprises a firstcombination of an RGD-PEG-polyamine conjugate with a polyamine polymer,such as PEI or a histidine-lysine copolymer, and a second combination ofthe said polymer mixture with a second mixture of dsRNAoligonucleotides. A solution comprising the polymer conjugate, orcomprising a mixture of a polymer conjugate with other polymer, lipid,or micelle such as materials comprising a ligand or a steric polymer orfusogen, is mixed with a solution comprising the nucleic acid, in oneembodiment an siRNA targeted against specific genes of interest, indesirable ratios to obtain nanoparticles that contain siRNA.

In this embodiment, nanoparticles are formed by layered nanoparticleself-assembly comprising mixing the polymer conjugate and the nucleicacid. Non-covalent electrostatic interactions between the negativelycharged nucleic acid and the positively charged segment of the polymerconjugate drive the self-assembly process that leads to formation ofnanoparticles. This process involves simple mixing of two solutionswhere one of the solutions containing the nucleic acid is added toanother solution containing the polymer conjugate followed by orconcurrent stirring. In one embodiment, the ratio between the positivelycharged components and the negatively charged components in the mixtureis determined by appropriately adjusting the concentrations of eachsolution or by adjusting the volume of solution added. In anotherembodiment, the two solutions are mixed under continuous flow conditionsusing mixing apparatus such as static mixer (FIG. 26). In thisembodiment, two or more solutions are introduced into a static mixer atrates and pressures giving a ratio of the solutions, where the streamsof solutions get mixed within the static mixer. Arrangements arepossible for mixers to be arranged in parallel or in series.

E. Combined Formulation and Electric Field

For certain applications, RNAi may be administered with or withoutapplication of an electric field. This can be used, for example, todeliver the RNAi molecules of the invention via direct injections into,for example, tumor tissue and directly into or nearby an angiogenictissue or a tissue with neovasculature. The siRNA may be in a suitablepharmaceutical carrier such as, for example, a saline solution or abuffered saline solution. The present invention, thus generallydescribed, will be understood more readily by reference to the followingexamples, which are provided by way of illustration and are not intendedto be limiting of the present invention.

EXAMPLES Example 1 Tumor Growth Inhibition with Combination Use of siRNADuplexes Targeting VEGFR2 and Bevacizumab (Avastin) Against VEGF

Material and Methods.

-   Reagents: Avastin, monoclonal antibody against VEGF (25 mg/ml,    Genetech); siRNA against mouse VEGFR2, sequence ((a)    AAGCTCAGCACACAGAAAGAC (SEQ ID NO: 247); (b) AATGCGGCGGTGGTGACAGTA)    (SEQ ID NO: 248); siRNA against luciferase (Qiagen); Avertin made of    1.5 gram 2,2,2,Tribromoethanol and 1.5 ml t-amyl alcohol (Cat#    T4840-2, Cat# 24048-6, Aldrich) in 100 ml distill water, St. Louis,    Mo.]. Mice: athymus female nude mice, 5 to 6 weeks old, were    purchased from TACONIC and housed conventionally. All investigations    followed guidelines of the Committee on the Care of Laboratory    Animals Resources, Commission of Life Sciences, National Research    Council. The animal facility of Biomedical Research Institute in    Rockville Md. is fully accredited by the American Association of    Laboratory Animal Care. Cells: Colon carcinoma cell line, DLD-1    (CCL-221, ATCC) was grown in RPMI 1640 medium with 2 mM L-glutamine,    1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM    sodium pyruvate, 10% fetal bovine serum.-   Procedure: 1) DLD-1 cells near confluence were harvested and    resuspended in serum-free RPMI medium. 2) Mice were anaesthetized    with Avertin, 0.4 ml/mouse i.p. 3) 100 million cells in 0.1 ml    serum-free RPMI medium were injected into mice back s.c. on the left    flank for establishment of xenograft tumor model. 4) 5 days after    inoculation of tumor cells, sizes of growing tumors were measured    with a caliper. Mice were then randomly grouped into 6 groups, 8    mice per group. 5) Experiment design and dosing scheme were as    following. 6) SiRNA was delivered through tail-vein injection after    been formed a complex with RPP.

TABLE I Regimen of anti-angiogenesis siRNA/mAb treatment of DLD-1 tumormodel Group Day-5 Day-6 Day-8 Day-9 Day-11 Day-12 Day-14 Day G1 -Luc-siRNA siRNA siRNA siRNA siRJ G2 - Avastin Avastin Avastin AvastinAvastin G3 - VEGFR2-siRNA siRNA siRNA siRNA siRJ G4 - Mouse-IgG + VEGFR2Mouse-IgG siRNA Mouse-IgG siRNA Mouse-IgG siRNA Mouse-IgG siRJ G5 -Avastin + Luc-siRNA Avastin siRNA Avastin siRNA Avastin siRNA AvastinsiRJ G6-Avastin + VEGFR2-siRNA Avastin siRNA Avastin siRNA Avastin siRNAAvastin siRJResults and Discussion

-   1. RGD ligand-directed nanoparticle carrying VEGF R2 specific siRNA    dsRNA itself was able to achieve tumor growth inhibition after    repeated systemic intravenous administrations (FIG. 1) with only 2    mg/kg siRNA.-   2. This siRNA-based antiangiogenesis activity (Targeting VEGF R2)    was able to enhance the VEGF monoclonal antibody (mAb)-mediated    antiangiogenesis, resulting further tumor growth inhibition (FIG.    1).-   3. Although many biochemical pathways may be critical independently    or collectively for malignant neoplasia, it is widely acknowledged    that neo-angiogenesis is critical for growth of many types of    cancers. Therefore neo-angiogenesis process has naturally become one    of the most targeted areas for tumor drug development. Among many    factors involved in neo-angiogenesis VEGF is believed an important    controlling factor and one that can enable a therapeutic effect    giving clinical benefit as achieved by Avastin, a monoclonal    antibody against VEGF and demonstrated to inhibit growth of colon    cancer. The combination of NV targeted VEGF siRNA gene inhibition    shows evidence of being additive with an antagonist therapeutic when    administrated in a combination protocol.-   4. The example shows the great benefit of using gene inhibitors and    with antagonists such as mAb inhibitors in the same treatment    regimen to improve the therapeutic outcome.-   5. RT-PCR results directly demonstrated that the VEGFR2 mRNA level    in the tumor samples treated with the nanoparticle delivered VEGFR2    specific siRNA has dramatically down regulated, comparing to the    expression levels in the untreated tumors (FIG. 2).-   6. Immunohistologicalchemistry (IHC analysis) indicated that the    protein level knockdown has occurred for VEGFR2 and CD31 which is a    marker for neo vasculature (FIG. 3).-   7. The ELISA measurement for IFN-alpha activity in the blood samples    from nanoparticle/VEGFR2 siRNA treatment alone, and in combination    with Avastin, did not show any significant (detectable) upregulation    or induction of IFN activity (FIG. 4), indicating that the    inhibition of NV and inhibition of tumor growth was not due to a    non-specific interferon-mediated effect.    Summary

This example illustrates the present invention as it providescompositions and methods for treatment of cancer with small interferingdsRNA oligonucleotide gene inhibitors in combination with monoclonalantibodies (mAb) in the same therapeutic regimen, where the agentsinhibit both expression of disease-causing genes and biologicalfunctions of disease-causing proteins, resulting in enhanced therapeuticefficacy. In addition, the example provides illustration ofsimultaneously blocking both the ligand (e.g. VEGF) and its receptor(e.g. VEGF R2) to provide effective inhibition of a particularbiological pathway (e.g. angiogenesis pathway) thereby providing atreatment inhibiting disease progression. Therefore, combinedapplications of inhibitory activities contributed by two potent andtargeted biological inhibitors, RNAi and mAb, is a means for effectivetreatment of NV diseases including cancer.

Example 2 siRNA-Mediated VEGF Silencing In Vitro and In Vivo

Human VEGF 165 specific siRNA was transfected into the MCF-7/VEGF165cell (hVEGF over-expression) by Lipofectamine resulting in knockdown ofhVEGF mRNA in the cell (FIG. 2 upper panel). Using an electroporationprocedure, MCF-7/VEGF165 cells were transfected by hVEGF165 specificsiRNA, resulting in down regulation of hVEGF expression as determinedwith an ELISA analysis.

When the hVEGF165 specific siRNA were delivered through intratumoraladministration repeatedly, the growth of MCF-7/VEGF165 cell inducedxenograft tumors were significantly inhibited. This inhibition wasfurther validated when the tumor tissues were collected for testinghVEGF mRNA expression which resulted in significant down regulationbased on RT-PCR analysis. Using the same hVEGF165 specific siRNA, we arealso able to demonstrate tumor growth inhibition with head and necksquamous cell carcinoma (HNSCC, 1483 cell) xenograft model, through 5repeated administrations intratumorally with seven day interval. Eachinjection requires 10 μg of the specific siRNA in 15 to 30 μL of RNAsefree aqueous solution.

Example 3 25 Basepair Blunted Double-Stranded siRNA is More Potent thanRegular 19 Basepair siRNA with 3′-Overhangs in Silencing Target GeneExpression

In one of in vitro siRNA transfection studies, the MCF/165 breast cancercells overexpressing human VEGF (hVEGF) were transfected with either 25basepair blunted double-stranded siRNA (hVEGF-25-siRNA-a,hVEGF-25-siRNA-b, hVEGF-25-siRNA-c, Luc-25-siRNA) or 19 basepair siRNAwith 3′ overhangs (hVEGF-siRNA-a, GFP-siRNA-a) using an electroporationmediated transfection method. 4×10⁶ MCF7/165 cells were resuspended in200 μl of siPORT siRNA Electroporation Buffer (Ambion) mixed with 5 μgsiRNA and then subjected to electroporation treatment using an ElectroSquare Porator ECM830 (BTX, Fisher Scientific). The parameters for theelectroporation are: voltage 500 v; duration of pulse 60 μs; pulsenumber 2; pulse interval 1 second. The transfected cells were seeded to24-well plate at a density of 5×10⁴ cell/well and cultured at 37° C.incubator with 5% CO₂. At 24 hours post transfection, the culture mediafrom each well were harvested and the concentration of human VEGFprotein in the media was measured using a commercial hVEGF ELISA kit(R&D Systems).

We observed a significant stronger hVEGF target gene inhibition in 25basepair hVEGF-siRNA treated cells than that in regular 19 basepairhVEGF siRNA treated cells. There was a more than 75% reduction of thesecreted hVEGF protein in the culture media of cells transfected with 25basepair blunted double-stranded hVEGF-25-siRNA molecules at 24 hourspost siRNA transfection, compared to an about 60% hVEGF proteinreduction observed in cells transfected with regular 19 basepairhVEGF-siRNA-a. Both non-specific sequence control 25 basepair blunteddouble-stranded siRNA (Luc-25-siRNA) or regular GFP-siRNA with3′-overhangs did not affect VEGF expression under the same siRNAtransfection condition (FIG. 7).

Example 4 25 Basepair Blunted Double-Stranded siRNA Mediated anElongated Target Gene Silencing

In another one of in vitro siRNA transfection studies, the MCF/165breast cancer cells overexpressing human VEGF (hVEGF) were transfectedwith either 25 basepair blunted double-stranded siRNA (hVEGF-25-siRNA-a,hVEGF-25-siRNA-b, hVEGF-25-siRNA-c, Luc-25-siRNA) or 19 basepair siRNAwith 3′ overhangs (hVEGF-siRNA-a, GFP-siRNA-a) using an electroporationmediated transfection method. 4×10⁶ MCF7/165 cells were resuspended in200 μl of siPORT siRNA Electroporation Buffer (Ambion) mixed with 2 μgor 5 μg siRNA and then subjected to electroporation treatment using anElectro Square Porator ECM830 (BTX, Fisher Scientific). The parametersfor the electroporation are: voltage 500 v; duration of pulse 60 μs;pulse number 2; pulse interval 1 second. The transfected cells wereseeded to 24-well plate at a density of 5×10⁴ cell/well and cultured at37° C. incubator with 5% CO₂. At 24, 48, 72, 96, and 120 hours posttransfection, the culture media from each well were harvested andreplaced with fresh culture media. The concentration of human VEGFprotein in the media harvested at various time points was measured usinga commercial hVEGF ELISA kit (R&D). The siRNA mediated hVEGF knockdownwas normalized with the hVEGF protein levels measured in cells treatedwith respective non-specific control siRNA.

We observed a significant stronger and elongated hVEGF target geneinhibition in 25 basepair hVEGF-siRNA treated cells than that in regular19 basepair hVEGF siRNA treated cells at every time points tested. At120 hours post siRNA treatment, there was still a more than 60%reduction of the secreted hVEGF protein in the culture media of cellstransfected with 5 μg of 25 basepair blunted double-strandedhVEGF-25-siRNA molecules, compared to an less than 20% hVEGF proteinreduction observed in cells transfected with 5 μg of regular 19 basepairhVEGF-siRNA-a (FIGS. 8 and 27). We also observed a dose-dependent siRNAmediated hVEGF gene inhibition for the 25 basepair blunteddouble-stranded hVEGF-25-siRNA molecules.

Base on our observation, the 25 basepair blunted double-stranded hVEGF-25-siRNA molecules not only gives a stronger target VEGF geneinhibition, but also results in a significant longer duration ofeffective target VEGF gene inhibition. For example, compared to only 48hours of more than 60% hVEGF protein reduction achieved using 5 μg ofregular 19 basepair hVEGF siRNA, at least 120 hours of more 60%reduction of protein was achieved using 25 basepair blunteddouble-stranded hVEGF siRNA. Therefore, the 25 basepair blunteddouble-stranded siRNA are more potent target gene inhibitor that canlead to more significant therapeutic efficacy.

Additional information include (1) Luc-25-siRNA has the sequence of(sense strand 5′-rGGAACCGCUGGAGAGCAACUGCAUA-3′ (SEQ ID NO: 249) andantisense strand 5′-rCCUUGGCGACCUCUCGUUGACGUAU-3′) (SEQ ID NO: 250); (2)hVEGF-siRNA-a has the sequence of (sense strand,5′-rUCGAGACCCUGGUGGACAUdTT-3′ (SEQ ID NO: 251) and antisense strand,5′-rAUGUCCACCAGGGUCUCGAdTT-3′) (SEQ ID NO: 252); (3) GFP-siRNA has thesequence of (sense strand, 5′-rGCUGACCCUGAAGUUCAUCdTT-3′ (SEQ ID NO:253) and (antisense strand, 5′-rGAUGAACUUCAGGGUCAGCdTT-3′) (SEQ ID NO:254); (4) hVEGFR2-25-siRNA-c: 5′-r(CCAAGUGAUUGAAGCAGAUGCCUUU)-3′ (SEQ IDNO: 255).

Example 5 25 Base Pair Blunted Double-Stranded siRNA EfficientlyKnockdown Human VEGFR1 and VEGFR2 Expression In Vitro

Three 25 base pair blunted double-stranded siRNA specifically targetinghuman VEGFR1 were transfected in HUVEC cells through electroporation.The membrane bound VEGFR1 and free extra cellular fragment of VEGFR1were measured 96 hours post transfection, through ELISA assay. TheVEGFR1-siRNA duplex a demonstrated the strongest VEGFR1 silencingactivity, compared to two other siRNA, for both cell lysate protein andthe free fragments in the cell culture supernatant solution (FIGS. 9 and10). When using the same approach to evaluate three 25 base pair blunteddouble-stranded siRNA targeting human VEGFR2 gene, we found all threeduplexes exhibiting potent silencing activity at both 24 hours and 72hours post transfection time points (FIG. 11).

Example 6 25 Basepair Blunted Double-Stranded siRNA Mediated StrongAnti-Tumor Efficacy in MDA-MB-435 Xenograft Tumor

25 basepair long siRNA duplex targeting human VEGF 165 gene sequencedemonstrated strong activity of tumor growth inhibition, after threerepeated intratumoral administrations of 10 μg every 5 days (FIG. 12),using a MDA-MB-435 breast carcinoma cell line known having highexpression of VEGF and bFGF proteins.

Example 7 siRNA Cocktail Targeting VEGF, VEGFR1 and VEGFR1 Genes is MorePotent than any of the Individual siRNA Targeting Only on of Those Genesin HSV Induced Ocular Neovascularization Model

Recently, we demonstrated that the siRNA cocktail containing siRNAtargeting VEGF, VEGFR1 and VEGFR2 can achieve more potentantiangiogenesis efficacy than only targeting one of those genes, at thesame dosage, in the animal disease model. Using this approach, we areable to efficiently block the VEGF pathway which plays the key role forpathological angiogenesis (FIG. 13).

HSV DNA that contains abundant potentially bioactive CpG-containingmotifs can induce the potent angiogenesis factor vascular endothelialgrowth factor (VEGF) and that neutralization of VEGF with antibodyminimized HSV-induced angiogenesis. A convenient model was alsoestablished in which bioactive CpG containing oligodeoxynucleotides(ODNs) were also shown to induce neovascularization via the induction ofVEGF. This model is used in the present study to evaluate thetherapeutic potential of RNA interference (RNAi) to suppress VEGFexpression and responsiveness.

Material and Methods

Reagents

Phosphorothioate ODNs were kindly provided by Dennis M. Klinman(Biologies Evaluation and Research, Food and Drug Administration,Washington, D.C.). The sequences of stimulatory ODNs used in this studywere: 1466, TCAACGTTGA (SEQ ID NO: 256), and 1555, GCTAGACGTTAGCGT (SEQID NO: 257). Subsequent studies were performed using an equimolarmixture of ODNs 1466 and 1555. Molecular Design of Gene Targets andsiRNA Three mVEGF pathway factors, mVEGF A and two mVEGF receptors(mVEGFR1 and mVEGFR2), were targeted by RNAi. For each gene target, twotarget sequences were assigned at different locations on the same mRNA.siRNA were designed correspondent to the above target sequences. ThesesiRNA were designed according to the guideline proposed by Tuschl. 14,15The designed siRNA (duplexes of sense and anti-sense strands) weresynthesized by Qiagen (Valencia, Calif.). All siRNA were 21-nucleotideslong doublestranded RNA oligos with a two nucleotide (TT) overhang atthe 3′-end. The targeted sequences of mVEGFA were (a)AAGCCGTCCTGTGTGCCGCTG (SEQ ID NO: 258) and (b) AACGATGAAGCCCTGGAGTGC(SEQ ID NO: 259).

The targeted sequences of mVEGFR1 were: (a) AAGTTAAAAGTGCCTGAACTG (SEQID NO: 260) and (b) AAGCAGGCCAGACTCTCTTTC (SEQ ID NO: 261). The targetedsequences of mVEGFR2 were (a) AAGCTCAGCACACAGAAAGAC (SEQ ID NO: 262) and(b) ATGCGGCGGTGGTGACAGTA (SEQ ID NO: 263). The synthesis of unrelatedsiRNA controls, two target sequences each for LacZ and fireflyluciferase were used. They were LacZ (a) AACAGTTGCGCAGCCTGAATG (SEQ IDNO: 264) and (b) AACTTAATCGCCTTGCAGCAC (SEQ ID NO: 265), Luc (a)AAGCTATGAAACGATATGGGC (SEQ ID NO: 266) and (b) AACCGCTGGAGAGCAACTGCA(SEQ ID NO: 267). Subsequent studies were conducted using an equimolarmixture of a and b for individual siRNA. Mice Female BALB/c mice (H-2d),5 to 6 weeks old, were purchased from Harlan Sprague-Dawley(Indianapolis, Ind.) and housed conventionally. All investigationsfollowed guidelines of the Committee on the Care of Laboratory AnimalsResources, Commission of Life Sciences, National Research Council. Theanimal facilities of the University of Tennessee (Knoxville, Tenn.) arefully accredited by the American Association of Laboratory Animal Care.

Virus HSV-I strain RE (kindly provided by Dr. Robert Lausch, Universityof Alabama, Mobile, Ala.) was used in all of the procedures. Virus wasgrown in Vero cell monolayers (catalog no. CCL81; American Type CultureCollection, Manassas, Va.), titrated, and stored in aliquots at −80° C.until used.In Vitro Efficacy of siRNA

To test the efficacy of RNAi in vitro, the following cell lines wereused. RAW264.7 gamma NO cells (CRL-2278, ATCC) were used to test theefficiency of siVEGFA specific knockdown of VEGFA gene that isspontaneously expressed in these cells. The cells were plated in asix-well plate in RPMI with 10% fetal bovine serum overnight at 37° C.in 5% CO₂. One day after cell plating, the cells were transfected withdifferent concentrations of siVEGFA or siLuc (at 0, 0.1, 0.5, 1.0, or2.0 μg/2 ml/well, respectively) using Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.). Twenty-four hours later RNA from these cells wasextracted for reverse transcriptase-polymerase RNA Extraction andRT-PCR). SVR cells (CRL-2280, ATCC) were used to test the efficiency ofsiVEGFR1-specific knockdown of VEGFR1 gene that is constitutivelyexpressed on these cells. The cells were plated in a six-well plate inDulbecco's modified Eagle's medium with 5% fetal bovine serum overnightat 37° C. in 5% CO₂. One day after cell plating, the cells weretransfected with different concentrations of siVEGFR1 or siLuc (at 0,0.1, 0.5, or 1.0 μg/2 ml/well, respectively) using Lipofectamine 2000.Forty-eight hours later RNA from these cells was extracted for RSPCR todetect VEGFR1 (see RNA Extraction and NATemplate-Specific PCR) (RS-PCR).The 293 cells (CRL-1573, ATCC) were used to transfect withmVEGFR2-expressing plasmid for the detection of knockdown of exogenousmVEGFR2. The cells were plated in a six-well plate in Dulbecco'smodified Eagle's medium with 5% fetal bovine serum overnight at 37° C.in 5% CO₂. One day after cell plating, the cells were cotransfected withplasmid pCI-VEGFR2 (0.2 μg/2 ml/well) and siVEGFR2 (a, b, a & b), orsiLuc (0, 0.1, 0.5, or 1.0 μg/well, respectively) using Lipofectamine2000. Forty eight hours later RNA from these cells was extracted forRS-PCR to detect VEGFR2.

Corneal Micropocket Assay

The corneal micropocket assay used in this study observed the generalprotocol of Kenyon and colleagues. Pellets for insertion into the corneawere made by combining known amounts of CpG ODNs, sucralfate (10 mg,Bulch Meditec, Vaerlose, Denmark), and hydron polymer in ethanol (120mg/1 ml ethanol; Interferon Sciences, New Brunswick, N.J.), and applyingthe mixture to a 15 mm² piece of synthetic mesh (Sefar America, Inc.,Kansas City, Mo.). The mixture was allowed to air dry and fibers of themesh were pulled apart, yielding pellets containing CpG ODNs. Themicropocket was made under a stereomicroscope (Leica Microsytems,Wetzlar, Germany) (four eyes per group) and pellets containing CpG ODNswere inserted into the micropocket. Angiogenesis was evaluated at days 4and 7 after pellet implantation by using calipers (Biomedical ResearchInstruments, Rockville, Md.) with a stereomicroscope. The length of theneovessels originated from the limbal vessel ring toward the center ofthe cornea and the width of the neovessels presented in clock hours weremeasured. Each clock hour is equal to 30° at the circumstance. Theangiogenic area was calculated according to the formula for an ellipse.

In Vivo Delivery of siRNA

Limbus was monitored at both day 4 and 7 after pellet implantation.Significant inhibition of corneal neovascularization resulted with allthree test siRNA compared to those given control siLacZ at day 4 afterpellet implantation (p<0.05). The combination of the three tested siRNAwas the most effective inhibitor, providing an approximately 60%reduction in neovascularization (p<0.01).

Inhibition of CpG-Induced Neovascularization by Systemic Delivery ofsiRNA Targeting VEGF-Pathway Genes.

To test the anti-angiogenic effect of targeted individual siRNA and theefficiency of systemic siRNA delivery, mice with CpG ODN-containingmicropockets were given a single dose i.v. of 40 μg siRNA containingeither siVEGFA, siVEGFR1, siVEGFR2, a mix of the three, or controlsiLacZ 6 and 24 h post pellet implantation. In these experiments apolymer (“Targetran”) was used that was shown in previous studies ontumor angiogenesis to facilitate extravascular delivery of siRNA. At day4 and 7 after pellet implantation, the extent of angiogenesis wasmeasured. All reagents used individually induced significant inhibitionof neovascularization compared to the siLacZ treated group at day 4after pellet implantation (p<0.05). As observed with localadministration, the mix of the three test reagents provided the mosteffective inhibition (average 40% inhibition, p<0.01). In additionalexperiments, the function of the polymer vehicle was evaluated bycomparing the anti-neovascularization activity of the test mix suspendedin polymer or given in PBS. These experiments revealed that the use ofthe polymer vehicle resulted in more effective anti16 neovascularizationthan was evident when the PBS vehicle was used although result was onlysignificant at the early test period (p<0.05). The results demonstratethat ocular neovascularization can be controlled by the i.v.administration of siRNA that target the VEGF system genes and that theuse of the RGD-mediated dsRNA nanoparticle delivery enhanced theefficacy of the therapeutic effect.

To determine the efficient anti-angiogenic dose of siRNA in systemicdelivery, mice with CpG ODN-containing micropockets were given a singledose i.v. of 10, 20, 40, 80 μg siRNA containing a mix of the siVEGFA,siVEGFR1 and siVEGFR2, or control siLuc with TargeTran vehicle at 6 and24 h post pellet implantation. A administration of siRNA inhibited CpGinduced angiogenesis in a dose dependent manner.

Therapeutic Application of siRNA Against VEGF-Pathway Genes in the HSKModel.

Previous studies have shown that VEGF is the critical angiogenic factorfor induction of HSV specific angiogenesis in the HSK model. To evaluatewhether administration of siRNA targeting VEGF-pathway genes inhibitsthe development of HSK, the corneas of mice were scarified and infectedwith 1-105 HSV-1 RE. Then mice were given a single dose of 10 μg(subconjunctival injection for local delivery) or 40 μg (tail veininjection for systemic delivery) mix of siRNA (an equimolar mixture ofsiVEGFA, siVEGFR1, and siVEGFR2) with polymer vehicle at day 1 and 3after virus infection. As shown in FIG. 4, the angiogenesis and severityof HSK was significantly reduced in mice treated with siRNA targetingVEGF-pathway genes either locally or systemically compared to animalstreated with siLuc control (p<0.05). Whilst 80% of siLuc control treatedeyes developed clinically evident lesions (score 2 or greater at day 10p.i.), only 42% (local delivery) or 50% (systemic delivery) of eyestreated with siRNA targeting VEGF-pathway genes developed such lesions.In addition by day 10 p.i., the angiogenesis score was greater than 6 in9 of 12 control eyes, but only in 5 of 12 eyes of mice treated withsiRNA against VEGF-pathway genes by either local or systemic delivery.Taken together these results show that administration of siRNA againstVEGF-pathway genes reduced development of HSK via inhibition ofangiogenesis.

Example 8 siRNA Cocktail Targeting VEGF, VEGFR1 and VEGFR1 Genes VeryPotent Anti-Angiogenesis Agent in ROP Ocular Neovascularization Model

Neovascularization in the eye is associated with various disorders,often causing severe loss of vision and eventually blindness. Amongthese disorders, diabetic retinopathy (DR), age-related maculardegeneration (AMD), retinal vein occlusion (RVO) and retinopathy ofprematurity (ROP) are prevalent. The imbalance of stimulatory factorsand inhibitory factors result in NV (NV) and vascular endothelial growthfactor (VEGF) is the most important factor of stimulatory factors whichcause vascular permeability, dilation and endothelial cell migration,proliferation^([1]).

RNA interference (RNAi) is the process of sequence-specificpost-transcriptional gene silencing in a wide range of organisms,initiated by double stranded RNA (dsRNA) that is homologous in sequenceto the targeting gene. In this example, small interference dsRNAoligonucleotides (siRNA) targeting VEGF path ways were used toinhibition NV induced by oxygen-induced retinopathy.

Materials and Methods

The Design and Synthesis of siRNA

siRNA was kindly provided by Intradigm Corporation. Three mVEGF pathwayfactors, mVEGF A and two mVEGF receptors (mVEGFR1 and mVEGFR2) weretargeted by RNAi and called siMix. The siRNA targeting luciferase wascalled siLuc as control.

Mouse Model of Oxygen Induced Retinal Neovascularization

The model we used (Smith et al.^([2])) imitates retinopathy ofprematurity. On postnatal day seven (P7) the mice and their nursingmother were placed in an airtight incubator (own production) ventilatedby a mixture of oxygen and air to a final oxygen fraction of 75%±2%.Oxygen levels were checked at least 3 times a day. On P12 the mice werereturned to room air. On P17 the animals were sacrificed.

Mice

The C57BL/6 mice were purchased from center of experimental animal ofGuangzhou Medical college and Guangzhou University of traditionalChinese Medicine. All investigations followed guidelines of theCommittee on the Care of Laboratory Animals Resources, Commission ofLife Science, National Research Council.

In Vivo Delivery of siRNA

For local delivery, siMix (4 μg/2 μl per eye) was deliveredsubconjunctivaly or intravitreally in the left eye and siLuc (4 μg/2 μlper eye) in the right eye by a 32-gauge Hamilton syringe (Hamilton Co,Revo, Nev.) on P12 and P13 under deep anesthesia. For systemicadministration, siRNA (15 μg/50 μl per mouse) was mixed with theRGD-PEG-PEI polymer conjugate preparation (TargeTran) and deliveredintraperitoneally on P12 and P13.

Retinal Angiography^([4])

On P17 the animals were sacrificed by cardiac perfusion with a solutionof 50 mg/ml fluorescein-labeled dextran in sodium chloride as describedpreviously. Both eyes were enucleated and fixed for 0.5-1 h in 10%buffered formaldehyde at room temperature. The anterior segment was cutoff and the neurosensory retina carefully removed. The retina was cutradially and flat mounted in glycerin, photoreceptors facing downward. Acover slip was placed over the retina and sealed with nail polish.Retinal whole mounts were examined by fluorescence microscopy. The areasof retinal NV were measured by soft Image-Pro Plus (Media Cybernetics,USA).

Cryosection^([5])

Eyes were removed and frozen in optimal cutting temperature embeddingcompound (Miles Diagnostics). Ocular frozen sections (10 μm) werehistochemically stained with biotinylated GSA. Slides were incubated inmethanol/H₂O₂ for 10 min at 4° C., washed with 0.05 M Tris-bufferedsaline (TBS), pH 7.6, and incubated for 30 min in 10% normal bovineserum. Slides were incubated 1 h at 37° C. with biotinylated GSA andafter rinsing with 0.05 M TBS, they were incubated with avidin coupledto alkaline phosphatase (Vector Laboratories) for 45 min at roomtemperature. After being washed for 10 min with 0.05 M TBS, slides wereincubated with diaminobenzidine to give a brown reaction product andwere counterstained with eosin, mounted with Cytoseal. To performquantitative assessments, 10 μm serial sections were cut through entireeyes, and starting from the 1st section that contained iris andextending to the last section on the other side of the eye thatcontained iris, every tenth section was stained with GSA and total 15sections were stained. GSA-stained sections were examined with anmicroscope, and images were digitized using a digital color videocamera. Image-Pro Plus software (Media Cybernetics, Silver Spring, Md.,USA) was used to delineate GSA-stained cells on the surface of theretina and their area was measured. For local injection the totalmeasurements from each eye was used as a single experimental value. Forsystemic injection the mean of both eyes of a mouse was used as a singleexperimental value.

RT-PCR

On P14 and P17 the mouse were sacrificed and total RNA was extractedfrom retinae with TRIzol reagent (Invitrogen, USA). Total RNA wasquantified at a ratio of 260 to 280 nm and 2 ug RNA was converted tocDNA via cDNA synthesis Kit (Fermentas, USA). RT-generated cDNAsencoding VEGF, VEGFR1, VEGFR2 and b-actin (acting as a control of RNAintegrity and as an internal standard) were amplified with RT-PCR.Amplification of 1.5 ul of cDNA was performed with 1 ul sense andantisense and 2.5 U of Taq polymerase. The oligonucleotide primersequences are: mVEGF (430 bp) □ forward 5′—GAT GTC TAC CAG CGA AGC TACTGC CGT CCG—3′ (SEQ ID NO: 268) □ reverse 5′—GAA CAT CGA TGA CAA GCT TAGGTA TCG ATA caa get gcc teg cct tg—3′ (SEQ ID NO: 269) □ mVEGFR1 (404bp) □ forward 5′—GTCAGC TGC TGG GAC ACC GCG GTC TTG CCT—3′ (SEQ ID NO:270) □ reverse 5′—GAA CAT CGA TGA CAA GCT TAG GTA TCG ATA tag art gaagat tec gc—3′ (SEQ ID NO: 271) □ mVEGFR2 (485 bp) □ forward 5′-TGG CTGGTC AAA CAG CTC ATC-3′ (SEQ ID NO: 272) □ reverse 5′-CTC ATC CAA GGG CAATTC ATC-3′ (SEQ ID NO: 273) □□ β-actin (232 bp) □ forward 5′-CAT TGT GATGGA CTC CGG AGA CGG-3′ (SEQ ID NO: 274) □ reverse 5′-CAT CTC CTG CTG AAGTCT AGA GC-3′ (SEQ ID NO: 275).

ELISA for VEGF and VEGF 1, VEGFR2

On P14 and P17 the mouse were sacrificed and retinae were extractedquickly on ice. Samples were homogenized in a cell lysis buffer(Mammalian cell lysis Kit, Biotechnology Department Bio Basid Inc,Canada) and were subsequently centrifuged at 12,000 rpm for 30 min. Thesupernatants were analyzed for protein concentrations via the BCAprotein quantitative analysis Kit (Shenery Biocolor Bioscience &Technology Company, China). Samples were diluted to a finalconcentration of 1 mg/ml. Levels of VEGF, VEGFR1, and VEGFR2 weredetermined using the Quantikine M Murine VEGF, sVEGFR1, and sVEGFR2Immunoassay Kits, respectively (R&D Systems Inc., Minneapolis, Minn.).Six to 12 tissue samples were analyzed for each group and time point.

Results and Discussion

Fluorescein Angiographic Assessment of the Effect of siRNA on Retinal NV

The retinas of the normal P17 mouse had both superficial and deepvascular layers (joined by connecting vessels) that extended from theoptic nerve to the periphery. Retinas from P17 control mouse exposed tohyperoxia contained many neo vascular tufts (high fluorescein) extendingfrom the surface of the retina at the junction between the perfused andnonperfused retina. After subconjunctival injection of siLuc or siMixthe retinas also had many NV, the areas had no obvious differences.Whereas the areas of NV after intravitreal and intraperitoneal injectionof siMix were significant less than the control.

Assessment of Effects of siRNA by Histologic Quantitation of Retinal NV.

Retinal NV was also assessed histologically by measured the GSA-positivecells (neovascularization) anterior to the internal limiting membrane(ILM). Retinas of P17 normoxic mice had superficial, middle, deepvessels and contained no endothelial cells anterior to the ILM. Retinasof P17 mice subjected to hyperoxia contained multiple neovascular tuftson the surface with some extending into the vitreous. Retinas of micetreated with subconjunctival injection of siLuc or siMix had nodifference with the areas of NV. The areas of NV after intravitreal andintraperitoneal injection of siMix were obviously reduced compared tosiLuc injection.

Decreased Level of VEGF, VEGFR1, VEGFR2 mRNA after Intravitreal andIntraperitoneal Injection of siRNA

To address whether treatment of siRNA against VEGF pathway genes reducesthe levels the VEGF, VEGFR1, VEGFR2 mRNA, retinas were collected on P14and P17 after intravitreal and intraperitoneal injection of siRNA. ThemRNA levels were measured by RT-PCR. The expression of VEGF, VEGFR1,VEGFR2 mRNA were reduced in the retinas treated with siMix against VEGFpathway genes compared to the control retinas treated with siLuc(p<0.05).

Decreased Expression of VEGF, VEGFR1, VEGFR2 Protein Levels afterIntravitreal and Intraperitoneal Application of siRNA

To evaluate whether treatment of siRNA targeting VEGF pathway genesdiminishes the production of VEGF, VEGFR1, VEGFR2 protein, ELISA wasused to measure VEGF, VEGFR1, VEGFR2 protein in siRNA-treated retinas onP14 and P17. As shown in table 1 and 2, VEGF, VEGFR1, VEGFR2 proteinlevels were lower in those that received siMix compared controls givenwith siLuc.

Imbalance in the demand and supply of oxygen and nutrients is theinitiating factor of neovascularization which induces upregulation ofVEGF. VEGF is not only the potent mitogenic factor for endothelialcells; it also induces vascular permeability and dilation. Thesebiological activities are mediated by binding VEGF to high-affinitytransmembrane autophosphorylating tyrosine kinase receptors^([5]). Threedistinct VEGF receptors have been identified, namely VEGFR1 (fms-linetyrosine kinase-1 or Flt-1), VEGFR2 (kinase insert domain-containingreceptor or KDR) and VEGFR3 (fms-line tyrosine kinase-4 or Flt-4).VEGFR1 and VEGFR2 are predominantly expressed on vascular, VEGFR3 onlymphatic endothelium^([6]). Increased intravitreal and intraretinallevels of VEGF are associated with retinal neovascularization not onlyin animal model but also in patients with ischemic retinopathy. Thesedata suggest that VEGF signaling is a good target for treatment ofretinal neovascularization^([5]).

Oxygen-induced retinopathy in mice is widely recognized in the world.Eric AP et al^([7]) had reported that the mRNA levels of VEGF increaseddramatically between 6 to 12 h after hypoxia and remained elevated forseveral days and then decreased toward baseline with regression ofretinopathy. So we delivered siRNA on P12 and P13 in order to interferethe synthesize of VEGF before upregulation. The results of whole mountand cryosection had proved that the NV areas of retinas treated withintravitreal and intraperitoneal injection of siMix were less thaninjection of siLuc. That is siMix can obviously inhibit retinal NV. Toexplore the mechanism the retinal VEGF, VEGFR1 and VEGFR2 expressions onP14 and P17 were examined and the mRNA and protein levels were lowerthan the siLuc controls. These suggested it is through inhibiting VEGFpathway genes that siMix inhibit retinal NV.

However there is no distinct difference after subconjuctival injection,so it is concluded that it hadn't achieved the effective concentrationin local. After intravitreal injection there was high concentration ofsiRNA in vitreous and siRNA was transfected into retinal neovascularendothelial cells. But this injection can result in intraocularhemorrhage and endophthalmitis et al. Intraperitoneal injection isrelative safe and siRNA can be absorbed into blood through abundantceliac capillary. But siRNA are easily trapped in the nonspecigic organsincluding liver, lungs, and spleen. So how to enhance the transfectingefficiency of siRNA is very important. The vehicle TargeTran is composedof polyethylene imine (PEI), polyethylene glycol (PEG) andarginine-glycine-aspartate peptide sequences (RGD). PEI binds tonegative charges in phosphates of the siRNA. RGD motif has beenidentified as an integrin ligand of activated endothelial cells.Endothelial cells express a number of different integrins and integrinαvβ3 and α5β1 have been shown to be important during angiogenesis. Bothintegrins are the receptor for matrix proteins with an exposed RDGtripeptide moiety and are most prominent on activated endothelial cellsduring angiogenesis. Thus the application of Targetran can improve thetransfecting efficiency of siRNA. Kim B et al^([8]) had reported thesubconjunctival and intravenous injection of siMix can inhibit the CPGinduced the corneal neovascularization in mice. It may also apply toother neovascular diseases.

Example 9 Ligand Directed Nanoparticle siRNA can be SpecificallyDelivery into Tumor with Systemic Administration

This self-assembled siRNA nanoparticle has demonstrated theneovasculature targeting property when they were systemicallyadministrated through IV injection. The tumor specific targetingcapability of this siRNA nanoparticle system has been revealed with aLuciferase report gene system.

Example 10 Ligand Directed Nanoparticle siRNA is Potent Anti-Tumor AgentValidated in Mouse Syngenic Model (Neuroglioblastoma)

Tumor targeted delivery of siRNA using the targeted nanoparticle systemfrom iv administration is demonstrated using fluorescently labeled siRNApackaged in RGD-PEG-PEI nanoparticle.

Mouse Tumor Model

Female nude mice (6-8 weeks of age) were obtained from Taconic(Germantown, N.Y.), kept in filter-topped cages with standard rodentchow and water available ad libitum, and a 12 h light/dark cycle.Experiments were performed according to national regulations andapproved by the local animal experiments ethical committee. SubcutaneousN2A tumors were induced by inoculation of 1×10⁶ N2A-cells in the flankof the mice. At a tumor volume of approximately 0.5-1 cm³, mice receivednanoplexes or free siRNA by i.v. injection of a solution of 0.2 ml viathe tail vein. 40 μg fluorescently-labeled siRNA was injected in thefree form or as PEI- or RGD-PEG-PEI-nanoparticle. One hr afterinjection, tissues were dissected and examined with a dissectionmicroscope fitted for fluorescence. Microscopic examination of tissueswas performed with an Olympus SZX12 fluorescence microscope equippedwith digital camera and connected to a PC running MagnaFire 2.0 camerasoftware (Optronics, Goleta, Calif.). Pictures were taken at equalexposure times for each tissue.

Result: Intravenous injection by tail vein of free fluorescent labeledsiRNA did nor show any significant accumulation in lung, liver or tumortissue. SiRNA delivered using PEI-nanoparticle showed highestaccumulation in the lung tissue followed by liver and tumor. SiRNAdelivered using RGD-PEG-PEI nanoparticle showed highest level ofaccumulation in tumor tissue. There was a considerable reduction in lungaccumulation compared to PEI-nanoparticles and very little accumulationin liver. This experiment demonstrates tumor targeted delivery of siRNAusing RGD-PEG-PEI nanoparticle.

RNAi mediated inhibition of tumor angiogenesis and tumor growth werestudied using tumor targeted nanoparticle formulation containing siRNAtargeted against VEGFR2. Subcutaneous tumor bearing mice were treated byintravenous injection of the nanoparticle formulation at a dose of 40 μgsiRNA per injection. Injections were repeated every third day and growthof the tumor was evaluated and compared with animals treated withcontrol formulations. Inhibition of the angiogenesis was evaluated atthe end of the experiment.

Mouse Tumor Model

Female nude mice (6-8 weeks of age) were obtained from Taconic(Germantown, N.Y.), kept in filter-topped cages with standard rodentchow and water available ad libitum, and a 12 h light/dark cycle.Experiments were performed according to national regulations andapproved by the local animal experiments ethical committee. SubcutaneousN2A tumors were induced by inoculation of 1×10⁶ N2A-cells in the flankof the mice. At a tumor volume of approximately 0.5-1 cm³, mice receivednanoplexes or free siRNA by i.v. injection of a solution of 0.2 ml viathe tail vein. The nanoparticle formulations containing siRNA wereprepared by simple mixing of siRNA solutions with polymer solution atgiven N/P ratio.

For the tumor growth inhibition studies, the experiment was started whenthe tumors became palpable, at 7 days after inoculation of the tumorcells. Treatment consisted of 40 μg siRNA per mouse in RPP-nanoplexesevery 3 days intravenously via the tail vein. Tumor growth was measuredat regular intervals using a digital caliper by an observer blinded totreatment allocation. Each measurement consisted of tumor diameter intwo directions approximately 90 degrees apart. Tumor volume wascalculated as, 0.52×longest diameter×shortest diameter². At the end ofthe experiment, the animals were sacrificed and tumor tissue andsurrounding skin was excised and put on a microscopy glass slide. Tissueexamination for vascularization and angiogenesis was performed bymicroscopy using the Olympus microscope and camera equipment describedabove for fluorescent tissue measurements. Tissue was trans-illuminatedto visualize blood vessels in the skin and a digital image was taken andstored as described above. Tissue was snap frozen immediately thereafterfor Western blotting.

Result: Significant tumor growth inhibition was observed for animalstreated with nanoparticle formulations containing VEGFR2 siRNA. Animalstreated with non-specific siRNA did not show any substantial tumorgrowth inhibition compared to untreated animals. Significant inhibitionof blood vessel growth was observed around the tumor tissue indicatingthe inhibition of angiogenesis in the VEGFR2-siRNA treated mice. Micetreated with control siRNA showed similar blood vessel growth as theuntreated animals. Western blot analysis of the tumor lysate collectedfrom animals of different treatment groups showed substantial reductionof VEGFR2 in VEGFR2-siRNA treated animals whereas no reduction in VEGFR2was observed in control siRNA treated animals. These experiments clearlydemonstrate the delivery of siRNA into tumor tissue from intravenousadministration. Effectiveness of VEGFR2 siRNA packaged in theRGD-PEG-PEI nanoparticle to inhibit tumor growth in renal cell carcinomamodel was studied using a 786-O xenograft tumor model. An experimentalprocedure similar to example 7 was used in this study. Briefly, femalenude mice (6-8 weeks of age) were obtained from Taconic (Germantown,N.Y.). Subcutaneous 786-O tumors were induced by inoculation of 5×10⁶786-O cells in the flank of the mice. At a tumor volume reachedapproximately 100 mm³, treatment was started by i.v. injection of asolution of VEGFR2-siRNA in RGD-PEG-PEI nanoparticle via the tail vein.Control treatment groups received nanoparticles containing non-specificsiRNA or saline. Treatment was repeated for several days with injectionsevery three days. Tumor volume was measured once every three days asdescribed in example 7. Result: Significant tumor growth inhibition wasobserved for animals treated with VEGFR2-siRNA nanoparticle formulation.No significant tumor growth inhibition was observed for animals treatedwith control siRNA nanoparticles. This experiment demonstrates that theVEGFR2 siRNA delivered by tumor targeted nanoparticle formulation canachieve tumor growth inhibition.

The ligand directed siRNA nanoparticles were systemically administratedinto a C57 mouse model with subcutaneous inoculation of N2A glioblastomacells for evaluation of the impact of VEGF knockdown on the tumorangiogenesis activity. Female nude mice (6-8 weeks of age) were obtainedfrom Taconic (Germantown, N.Y.), kept in filter-topped cages withstandard rodent chow and water available ad libitum, and a 12 hlight/dark cycle. The experiments were performed according to nationalregulations and approved by the local animal experiments ethicalcommittee. Subcutaneous N2A tumors were induced by inoculation of 1×10⁶N2A cells in the flank of the mice. At a tumor volume of 0.5-1 cm3, micereceived nanoplexes or free siRNA by i.v. injection of a solution of 0.2ml via the tail vein. The nanoplex solutions were prepared as above, atN/P ratio of 2. For tissue distribution experiments, 40 mg fluorescentlylabeled siRNA was injected in the free form or as P- or RPP-nanoplexes.One hour after injection, the tissues were dissected and examined with adissection microscope fitted for fluorescence. Microscopic examinationof tissues was performed with an Olympus SZX12 fluorescence microscopeequipped with digital camera and connected to a PC running MagnaFire 2.0camera software (Optronics, Goleta, Calif.). Pictures were taken atequal exposure times for each tissue.

In the co-delivery experiments, plasmid and siRNA were mixed in a 1:100molar ratio, respectively (40 mg pLuc with 13 mg siRNA), and in thesequential delivery experiments 40 mg plasmid was delivered first,followed by 40 mg siRNA 2 h later (1:300 molar ratio). The tissues weredissected, weighed and put in ice-cold reporter lysis buffer (Promega)in magnetic beads containing 2 ml tubes (Q-Biogene, Carlsbad, Calif.),24 h after injection of the nanoplexes. Tissues were homogenized with aFastprep FP120 magnetic homogenizer (Q-Biogene) and samples were assayedfor reporter enzyme activity using the luciferase assay system (Promega)on a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Inthe tumor growth inhibition studies, the experiment was started when thetumors became palpable at 7 days after inoculation of the tumor cells.Treatment consisted of 40 mg siRNA per mouse in RPP-nanoplexes every 3days intravenously via the tail vein. Tumor growth was measured atregular intervals using a digital caliper by an observer blinded totreatment allocation. Each measurement consisted of tumor diameter intwo directions of_(—)90_apart. Tumor volume was calculated as:0.52×longest diameter×shortest diameter².

At the end of the experiment, the animals were sacrificed and tumortissue and surrounding skin was excised and put on a microscopy glassslide. Tissue examination for vascularization and angiogenesis wasperformed by microscopy using the Olympus microscope and cameraequipment described above for fluorescent tissue measurements. Tissuewas trans-illuminated to visualize blood vessels in the skin and adigital image was taken and stored as described above. Tissue was snapfrozen immediately thereafter for western blotting.

Tumor growth inhibition by siRNA RPP-nanoplexes. Mice were inoculatedwith N2A tumor cells and left untreated or treated every 3 days by tailvein injection with RPP-nanoplexes with siLacZ or siVEGF R2 at a dose of40 mg per mouse. Treatment was started at the time-point that the tumorsbecame palpable (20 mm³). Only VEGF R2-sequence-specific siRNA inhibitedtumor growth, whereas treatment with LacZ siRNA did not affect tumorgrowth rate as compared with untreated controls (n=5).

Example 11 Ligand Directed Nanoparticle siRNA is Potent Anti-Tumor AgentValidated in Xenograft Tumor Model (Renal Carcinoma)

The ligand directed siRNA nanoparticles were also tested in a mousexenograft model with a renal carcinoma cell lines. The siRNAnanoparticles were manufactured with the same materials and sameprocedure, using the same route of delivery as that of Example 10. Fivetimes of repeated deliveries with three day intervals using 2 mg/kgdosage were carried out with six animals per cohort. The significanttumor growth inhibition was observed.

Example 12 Ligand Directed Nanoparticle siRNA is Potent Anti-Tumor AgentValidated in Xenograft Tumor Model (Colorectal Carcinoma)

Using siRNA nanoparticle targeting VEGFR2 gene, we further tested theantitumor efficacy in mouse xenograft model with a human colorectalcarcinoma cell line, DLD-1.

Material and Methods. Reagents: Avastin, monoclonal antibody againstVEGF (25 mg/ml, Genetech); siRNA against VEGFR2, sequence (Appendix 1);siRNA against luciferase (Qiagen); Avertin made of 1.5 gram2,2,2,Tribromoethanol and 1.5 ml t-amyl alcohol (Cat# T4840-2, Cat#24048-6, Aldrich) in 100 ml distill water, St. Louis, Mo.]. Mice:athymus female nude mice, 5 to 6 weeks old, were purchased from TACONIC( ) and housed conventionally. All investigations followed guidelines ofthe Committee on the Care of Laboratory Animals Resources, Commission ofLife Sciences, National Research Council. The animal facility ofBiomedical Research Institute in Rockville Md. is fully accredited bythe American Association of Laboratory Animal Care. Cells: Coloncarcinoma cell line, DLD-1 (CCL-221, ATCC) was grown in RPMI 1640 mediumwith 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10mM HEPES, and 1.0 mM sodium pyruvate, 10% fetal bovine serum.Reagents

Avastin, monoclonal antibody against VEGF (25 mg/ml, Genetech); siRNAagainst VEGFR2, sequence (Appendix 1); siRNA against luciferase(Qiagen); Avertin made of 1.5 gram 2,2,2,Tribromoethanol and 1.5 mlt-amyl alcohol (Cat# T4840-2, Cat# 24048-6, Aldrich) in 100 ml distillwater, St. Louis, Mo.]. Mice: athymus female nude mice, 5 to 6 weeksold, were purchased from TACONIC ( ) and housed conventionally. Allinvestigations followed guidelines of the Committee on the Care ofLaboratory Animals Resources, Commission of Life Sciences, NationalResearch Council. The animal facility of Biomedical Research Institutein Rockville Md. is fully accredited by the American Association ofLaboratory Animal Care. Cells: Colon carcinoma cell line, DLD-1(CCL-221, ATCC) was grown in RPMI 1640 medium with 2 mM L-glutamine, 1.5g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodiumpyruvate, 10% fetal bovine serum. Procedure: 1) DLD-1 cells nearconfluence were harvested and resuspended in serum-free RPMI medium. 2)Mice were anaesthetized with Avertin, 0.4 ml/mouse i.p. 3) 100 millioncells in 0.1 ml serum-free RPMI medium were injected into mice back s.c.on the left flank for establishment of xenograft tumor model. 4) 5 daysafter inoculation of tumor cells, sizes of growing tumors were measuredwith a caliper. Mice were then randomly grouped with 7 mice per group.Three different dosing regimen were applied: 1 mg/kg, 2 mg/kg and 4mg/kg respectively. Although the high dose at 4 mg/kg represented thestrongest anti-tumor activity, there is no significant difference amountthree treatment groups. Using a different comparison, it has been foundthat the high dose of the siRNA nanoparticle at 4 mg/kg exhibited astronger anti-tumor efficacy than that of 5 mg/kg Avastin treatment.

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TABLE B1-1 human VEGF specific siRNA sequences (25 basepairs with bluntends): VEGF-1, CCUGAUGAGAUCGAGUACAUCUUCA (SEQ ID NO: 276) VEGF-2,GAGUCCAACAUCACCAUGCAGAUUA (SEQ ID NO: 277) VEGF-3,AGUCCAACAUCACCAUGCAGAUUAU (SEQ ID NO: 278) VEGF-4,CCAACAUCACCAUGCAGAUUAUGCG (SEQ ID NO: 279) VEGF-5,CACCAUGCAGAUUAUGCGGAUCAAA (SEQ ID NO: 280) VEGF-6,GCACAUAGGAGAGAUGAGCUUCCUA (SEQ ID NO: 281) VEGF-7,GAGAGAUGAGCUUCCUACAGCACAA (SEQ ID NO: 282)

TABLE B1-3 human VEGFR1 specific siRNA sequences (25 base- pairs withblunt ends): VEGFR1- CAAAGGACUUUAUACUUGUCGUGUA (SEQ ID NO: 283) 1,VEGFR1- CCCUCGCCGGAAGUUGUAUGGUUAA (SEQ ID NO: 284) 2, VEGFR1-CAUCACUCAGCGCAUGGCAAUAAUA (SEQ ID NO: 285) 3, VEGFR1-CCACCACUUUAGACUGUCAUGCUAA (SEQ ID NO: 286) 4, VEGFR1-CGGACAAGUCUAAUCUGGAGCUGAU (SEQ ID NO: 287) 5, VEGFR1-UGACCCACAUUGGCCACCAUCUGAA (SEQ ID NO: 288) 6, VEGFR1-GAGGGCCUCUGAUGGUGAUUGUUGA (SEQ ID NO: 289) 7, VEGFR1-CGAGCUCCGGCUUUCAGGAAGAUAA (SEQ ID NO: 290) 8, VEGFR1-CAAUCAAUGCCAUACUGACAGGAAA (SEQ ID NO: 291) 9, VEGFR1-GAAAGUAUUUCAGCUCCGAAGUUUA (SEQ ID NO: 292) 10,

TABLE B1-5 human VEGFR2 specific siRNA sequences (25 base- pairs withblunt ends): VEGFR2- CCUCGGUCAUUUAUGUCUAUGUUCA (SEQ ID NO: 293) 1,VEGFR2- CAGAUCUCCAUUUAUUGCUUCUGUU (SEQ ID NO: 294) 2, VEGFR2-GACCAACAUGGAGUCGUGUACAUUA (SEQ ID NO: 295) 3, VEGFR2-CCCUUGAGUCCAAUCACACAAUUAA (SEQ ID NO: 296) 4, VEGFR2-CCAUGUUCUUCUGGCUACUUCUUGU (SEQ ID NO: 297) 5, VEGFR2-UCAUUCAUAUUGGUCACCAUCUCAA (SEQ ID NO: 298) 6, VEGFR2-GAGUUCUUGGCAUCGCGAAAGUGUA (SEQ ID NO: 299) 7, VEGFR2-CAGCAGGAAUCAGUCAGUAUCUGCA (SEQ ID NO: 300) 8, VEGFR2-CAGUGGUAUGGUUCUUGCCUCAGAA (SEQ ID NO: 301) 9, VEGFR2-CCACACUGAGCUCUCCUCCUGUUUA (SEQ ID NO: 302) 10,

TABLE B1-7 human EGF specific siRNA sequences (25 basepairs with bluntends): EGF-1, CAGGUAUUUCUUACUGGCUUCCAAA (SEQ ID NO: 303) EGF-2,GCUGGUGUCUCAGUGAUCAUGGAUU (SEQ ID NO: 304) EGF-3,CAGAUCUCGAUGGUGUGGGAGUGAA (SEQ ID NO: 305) EGF-4,GAUGGAGGUUCUGUCCACAUUAGUA (SEQ ID NO: 306) EGF-5,GCCAAGAGAUUAUUCUGGACUGAUA (SEQ ID NO: 307) EGF-6,CGAUGCCAAGCAGUCUGUGAUUGAA (SEQ ID NO: 308) EGF-7,UGGCAGGUGGUGAAGUUGAUCUAAA (SEQ ID NO: 309) EGF-8,GCGAAGCUUUCAUAUGCCCUCCUAU (SEQ ID NO: 310) EGF-9,UCUCUACGACUAAUCACCUACUCAA (SEQ ID NO: 311) EGF-10,CACUGCAGUCUUAUUUCCAAGUAAG (SEQ ID NO: 312)

TABLE B1-9 human EGFR specific siRNA sequences (25 basepairs with bluntends): EGFR-1, CACAGUGGAGCGAAUUCCUUUGGAA (SEQ ID NO: 313) EGFR-2,CGCAAAGUGUGUAACGGAAUAGGUA (SEQ ID NO: 314) EGFR-3,CGCAGCAUGUCAAGAUCACAGAUUU (SEQ ID NO: 315) EGFR-4,CAGAAGGAGGCAAAGUGCCUAUCAA (SEQ ID NO: 316) EGFR-5,GCCUAUCAAGUGGAUGGCAUUGGAA (SEQ ID NO: 317) EGFR-6,CCAAGUCCUACAGACUCCAACUUCU (SEQ ID NO: 318) EGFR-7,UCUCUGAGUGCAACCAGCAACAAUU (SEQ ID NO: 319) EGFR-8,CAGCUUCUUGCAGCGAUACAGCUCA (SEQ ID NO: 320) EGFR-9,GAAGCCAAGCCAAAUGGCAUCUUUA (SEQ ID NO: 321) EGFR-10,UACAGAAACGCAUCCAGCAAGAAUA (SEQ ID NO: 322)

1. A small interfering RNA (siRNA) molecule, wherein the siRNA isdouble-stranded and consists of a sense strand consisting of thesequence CACAGUGGAGCGAAUUCCUUUGGAA (SEQ ID NO: 313) and an anti-sensestrand consisting of the complement of CACAGUGGAGCGAAUUCCUUUGGAA (SEQ IDNO: 313) and wherein the siRNA reduces EGFR expression in a cell.
 2. ThesiRNA molecule of claim 1, comprising at least one nucleotide that ischemically modified.
 3. The siRNA molecule of claim 2, wherein the atleast one chemically modified nucleotide is a nucleotide comprising a2′-O-methyl ribose.
 4. The siRNA molecule of claim 2, wherein the atleast one chemically modified nucleotide is in the sequence in SEQ IDNO:
 313. 5. The siRNA molecule of claim 2, wherein the at least onechemically modified nucleotide is in the complement of the sequence inSEQ ID NO:
 313. 6. A composition comprising the siRNA molecule of claim1 and a pharmaceutically acceptable carrier.
 7. The composition of claim6, further comprising one or more additional nucleic acid molecules thatinduce RNA interference and decrease the expression of a gene ofinterest.
 8. The composition of claim 7, wherein at least one of the oneor more additional nucleic acid molecules decreases the expression of agene selected from the group consisting of VEGF, VEGFR1, VEGFR2, VEGFR3,PDGF, PDGFR-α, PDGFR-β, EGF, EGFR, RAF-a, RAF-c, AKT, RAS, NFkB, HIF,bFGF, bFGFR, Her-2, c-Met, c-Myc and HGF.
 9. The composition of claim 6,wherein the carrier is a nucleic acid delivery vehicle.
 10. Thecomposition of claim 9, wherein the nucleic acid delivery vehicle issynthetic.
 11. The composition of claim 10 wherein the synthetic nucleicacid delivery vehicle comprises a cationic polymer-siRNA complex. 12.The composition of claim 11, wherein the cationic polymer is ahistidine-lysine co-polymer.
 13. The composition of claim 11, whereinthe synthetic nucleic acid delivery vehicle further comprises ahydrophilic polymer.
 14. The composition of claim 13, wherein thehydrophilic polymer comprises a component selected from the groupconsisting of: polyethylene glycol, a polyacetal and a polyoxazoline.15. The composition of claim 10, wherein the synthetic nucleic acidvehicle further comprises a targeting moiety.
 16. The composition ofclaim 15, wherein the targeting moiety binds a tumor specific moleculeor an angiogenesis-specific molecule.
 17. The composition of claim 6comprising an additional therapeutic agent selected from the groupconsisting of: an anti-cancer agent, an anti-inflammatory agent and ananti-infective agent.
 18. The composition of claim 6 comprising anadditional anti-angiogenic agent.
 19. A 25 base pair, blunt-ended siRNAmolecule that is double-stranded and comprises a sense strand and anantisense strand, wherein the antisense strand consists of thecomplement of the sequence in SEQ ID NO: 313 and wherein the siRNAreduces EGFR expression in a cell.
 20. The siRNA molecule of claim 19,comprising at least one nucleotide that is chemically modified.
 21. ThesiRNA molecule of claim 20, wherein the at least one chemically modifiednucleotide is a nucleotide comprising a 2′-O-methyl ribose.
 22. ThesiRNA molecule of claim 20, wherein the at least one chemically modifiednucleotide is in the sense strand.
 23. The siRNA molecule of claim 20,wherein the at least one chemically modified nucleotide is in theantisense strand.
 24. A composition comprising the siRNA molecule ofclaim 19 and a pharmaceutically acceptable carrier.
 25. The compositionof claim 24, wherein the composition comprises a cationic polymer. 26.The composition of claim 25, wherein the cationic polymer is ahistidine-lysine co-polymer.
 27. The composition of claim 24, whereinthe composition comprises a hydrophilic polymer.
 28. The composition ofclaim 24, wherein the composition comprises a targeting ligand.
 29. AsiRNA molecule, wherein the siRNA is double-stranded and consists of asense strand consisting of the sequence CACAGUGGAGCGAAUUCCUUUGGAA (SEQID NO: 313), optionally with an overhang of one to four nucleotides andan anti-sense strand consisting of the complement ofCACAGUGGAGCGAAUUCCUUUGGAA (SEQ ID NO: 313), optionally with an overhangof one to four nucleotides and wherein the siRNA reduces EGFR expressionin a cell.